Rozprawy doktorskie na temat „Nano-formulations for drug delivery”

Overcoming the excellent barrier properties of the human skin represents the major challenge of this route of delivery. The Metered dose transdermal spray (MDTS®) is a technology developed by Acrux Ltd (AUS). This passive delivery system has the potential to avoid skin irritation. An informed choice of solvents is one of the strategies to design efficient transdermal formulations. Therefore, it is the aim of this thesis to develop optimal volatile formulations and to investigate the enhancement effects of solvents on drug permeation through the skin. A secondary objective is to evaluate the influence of solvent thermodynamic activity on drug permeation and the transport of solvent through skin on drug delivery. Miscibility, solubility, solvent uptake and residence time studies were conducted as a basis for formulation development. The effects of selected solvents on drug permeation were studied using a flow through diffusion cell design, under clinical dosing conditions of use. The influence of formulation related parameters such as solvent dose, supersaturation, combination of solvents and solvent thermodynamic activity, on the amount of ibuprofen permeated through human epidermis was further studied in vitro. The permeation of octyl salicylate, Propylene glycol and polyethylene glycol 200 was monitored by HPLC/UV, GC/MS and LC/MS, respectively, from selected volatile formulations. From these studies, it was found that ibuprofen permeation from all the residual phases studied was solvent dependent. Superior enhancement was obtained using polyethylene glycol 200 followed by propylene glycol, dipropylene glycol and transcutol®. The excipients in which ibuprofen had higher solubility showed a promotion of drug transport. Furthermore, concentration-dependent effect on ibuprofen permeation from solvents was observed. Significant improvements in the permeation of ibuprofen through human skin were achieved using combinations of solvents. The residual phase composed of polyethylene glycol: octyl salicylate (5:1 and 5:10 % (w/v)) were the best solvent/vehicle for ibuprofen permeation. The findings indicate that drug transport appears to be a function not only of the residual phase excipients, but also of the ultimate fate of the excipients after application. Finally, the reported findings demonstrate the potential of volatile formulations to optimise the efficiency of drug delivery to the skin.

Pulmonary and oral drug administrations are usually the preferred methods of delivery of active pharmaceutical ingredients.Generally,pulmonary drug formulations are more attractive compared to oral formulations since they consist of micron-sized powders with high surface area thus having faster onset of action,as well as minimizing the drug dosage and side effects.Oral insulin formulations,if achievable,would provide an alternative to injectable insulin,as the common drawbacks of injectable insulin are the multiple daily injections and the possibility of skin infections at the injection site. In this study,the feasibility of using dense gas particle processing techniques known as the Aerosol Solvent Extraction System (ASES),Gas Anti-Solvent (GAS)and High-Pressure Media Milling (HPMM)for pharmaceutical processing was assessed.The ASEStechnique,utilizing dense ethane,was employed to prepare insulin-lactose formulations for pulmonary administration whilst the GAS and ASES techniques,utilizing dense CO2,were employed to prepare microencapsulated formulations containing insulin and Eudragit?? S100 for oral administration.Furthermore,the HPMM technique,utilizing dense hydrofluocarbon (HFC)134a/227ea,was employed to prepare suspension Metered Dose Inhaler (MDI)formulations containing budesonide and various surfactants. The Fine Particle Fraction (FPF)of processed insulin without the presence of lactose was found to be 44%.In other words,44% of processed insulin delivered to the impactor stages (excluding the throat and neck)has aerodynamic diameter of less than 5??m.With the addition of lactose as carrier,the FPFof the insulin-lactose (1:1w/w)formulation increased to 64%.The increase in FPFwas attributed to the lower density of lactose particles compared to that of insulin particles to produce an intimate mixture with enhanced powder flowability and aerodynamic performance. Proteins for oral delivery should ideally be formulated with acid-resistant polymer as a protective coating to protect against enzymatic degradation in the stomach.Eudragit?? S100,which is insoluble or almost impermeable at pH 1-4and soluble at pH 5-7,was used to prepare oral insulin formulations.The insulin release at pH 3was sustained by the Eudragit?? S100coating and the encapsulation efficiency of insulin??Eudragit?? S100formulations varied between 6% and 24% depending on the initial drug to polymer ratio. One of the major therapies utilizing metered dose inhaler formulations in the treatment of asthma has been studied using the HPMM process.The HPMM process has been demonstrated to be an efficient milling process for the enhancement of the physical stability and aerodynamic performance of budesonide in HFC-134a/227ea propellant formulations.No significant change in physical stability was observed in the formulations for 2 weeks.

This study aims to compare the efficiency of conventional liposomes and surfactant-enriched vesicles (surfactosomes) using the hydrophilic drug salbutamol sulphate (SBS) and the hydrophobic drug beclometasone dipropionate (BDP) for pulmonary delivery via nebulisation. Initially liposomes and surfactosomes with or without cholesterol were prepared using thin film method and were compared for their VMD, span and drug entrapment. Their drug retention on extrusion through 5µm, 2µm, 1µm and 0.4µm polycarbonate membrane using mini-extruder was also studied. It was observed that liposomes were more stable than surfactosome. Particulate based proliposome technology was also used to study their potential for generating stable and inhalable dispersions. Mannitol was used as the carbohydrate carrier and on hydration; proliposomes and prosurfactosomes have generated liposomes and surfactosomes respectively. The VMD, span and zeta potential of the vesicles, and drug entrapment and drug retention on extrusion were studied. It was seen that lower proportions of SBS were entrapped using proliposome technology; hence, further extrusions through 5µm and 2µm were avoided. In vesicle with BDP, inclusion of cholesterol has decreased the drug entrapment and crystallisation of mannitol was observed. Nebulisation of liposomes and surfactosomes with and without cholesterol was studied using PARI LC sprint air jet nebuliser, Aeroneb pro and Beurer iH50 vibrating mesh nebulisers. Two stage (Twin) impinger was used to study the potential suitability of the generated vesicles for inhalation. VMD, span and zeta potential of vesicles before and after nebulisation was studied. BDP delivery and retention in both stages of the twin impinger was also studied. It was found that surfactosomes without cholesterol delivered maximum BDP to the twin impinger. Nebulisers suitable for all four formulations were also studied. Beurer iH50 delivered maximum BDP via liposomes with and without cholesterol, Aeroneb Pro delivered maximum BDP via surfactosomes with cholesterol to upper impinger while PARI LC sprint delivered maximum BDP via surfactosomes with cholesterol. VMD and span of aerosols generated from all three nebulisers were also studied. Stability of liposomes and surfactosomes prepared using proliposome technology was studied. VMD, span, zeta potential and BDP retention before and after spray drying and freeze drying were investigated. It was concluded that liposomes and surfactosomes were equally stable when spray drying was used whereas liposomes were more stable that surfactosomes when freeze drying was conducted. X-ray diffraction, scanning electron microscopy and transmission electron microscopy were used to analyse the characteristics of proliposomes and prosurfactosomes. A reduction in size and crystallinity was observed after spray drying and freeze drying of the formulations. Stability was also studied on storing proliposome and prosurfactosome in different environmental conditions like 5-6°C, room temperature and 40°C for a period of 3 months. It was concluded that both proliposomes and surfactosomes were most stable in 2-8°Cwhereas least stable in 40°C. Proliposomes were more stable than prosurfactosomes regardless of the storage temperature. Formulation and characterisation of novel prosurfactosomes and comparing it with conventional liposomes for pulmonary drug delivery is the novelty of this thesis.

The master thesis has investigated primarily on the synthesis of different polymeric NPs viz PLA (synthesized in house as well as commercial grade), PLGA (commercial) and PCL(synthesized in house, having different functionalities ?COOH, -PEG and their blends) employing flash nano-precipitation technique in a multi inlet vortex mixer (MIVM), previously optimized in the Morbidelli group at ETH. These NPs were characterized using DLS (Dynamic Light Scattering) and Zeta-Sizer to report the variation of the sizes and zeta potentials respectively of the NPs as a function of polymer molecular weight and initial concentration of polymer. The lowest possible sizes of the NPs were then selected for further studies as the overall motivation of the work is to synthesize NCs composed of primary particles and thereafter compare and contrast drug loading, encapsulation efficiencies and release kinetics of a model drug between the two. Ibuprofen (model drug) was loaded into the primary NPs using the MIVM setup, following which drug loading and encapsulation efficiencies were measured using High Performance Liquid Chromatography (HPLC). The release kinetics experiments were performed at 37°C and also studied at room temperature (25°C) and 45°C to evaluate the effect of temperature on release mechanism. The drug-loaded NPs were separated from the free drug in solution at different times using centrifugal filtration. The amount of drug released over time was measured by analyzing these supernatants using HPLC. The MIVM setup is found to produce stable polymeric NPs as small as 50nm and as large as 155nm depending on polymer concentration and nature of polymer. The results indicate that this setup is capable of producing drug loaded NPs with high drug loading efficiencies varying between 75% and 88% differing with polymers. This particular aspect has been established to be both reproducible and valid for a wide range of polymers through subsequent experiments. On the contrary, the release kinetics from almost all the different types of polymeric systems is slow; lasting over several days and moreover, it is not possible to release the entire loaded drug. It is claimed that either the chemical interaction of the polymers with ibuprofen or the location of the drug inside the polymeric NPs is the potential reason for extremely slow release kinetics. It is therefore suggested that further investigation is needed for the same system with another drug, having similar solubility parameters as ibuprofen to confirm the observed behaviour or even a completely different synthesis method for drug loaded polymeric NPs using ibuprofen to substantiate the observed results.

The primary objective of this research was to investigate the possible effects of selected liquid crystal (LC) forming surfactants, namely ArlacelTM 2121, CrodafosTM CES and BrijTM system (BrijTM S721/ BrijTM S2) and selected oils, namely, Arlamol™ PS15E, Crodamol™ OP, Arlamol™ HD on formulation properties. The effects of the different excipients were monitored using the formulations thermal properties, water holding ability and ability to promote the permeation of ibuprofen (IBU and caffeine (CAF)) across silicone membranes. The melting endotherms of the ternary formulations containing 10% w/w Arlamol™ PS15E or Crodamol™ OP, 10% w/w surfactant and 80% w/w water resulted in high melting endotherm (>55˚C). However, the inclusion of 10% w/w Arlamol™ HD in equivalent ternary formulations lowered the melting endotherm to 45-50 ˚C, suggesting a destabilising effect of this oil. In addition, increasing the surfactant content of ternary formulations from 5% w/w to 10% w/w reduced the evaporation time of free water by 10-20 min. However, this change in the water holding ability was not the same for all surfactants. The results ranked the water holding ability of the surfactants as the Brij™ system > CrodafosTM CES > Arlacel™ 2121. The permeation profiles of IBU and CAF across model membranes showed significant enhancement (p <0.05) for both drugs from saturated formulations containing Crodamol™ OP with the Brij™ surfactant system. This was attributed to the uptake of Crodamol™ OP into silicone membrane (22.88%) and the good solubility of both model actives in this oil. In addition, the permeation results suggest that the Brij™ system was interacting with membranes at a greater extent compared with other surfactants. The thesis also investigated the hydration characteristics of the human nail using the Confocal Raman spectroscopy (CRS). The results showed the nail to absorb significant amounts of water (~15% w/w) into the top 5 µm of the nail plate after 10 min of hydration. The absorbed water was lost from the nail in a quick manner of 30 min which agreed with previous reports. The CRS was also used to monitor in vivo deposition of IBU from saturated propylene glycol (PG) solutions under infinite (occluded) conditions. The results were consistent with previous work, showing IBU signal inside the skin to increase consequently with PG content in the applied formulation. These results indicated that CRS can be used as a valid in vivo technique to monitor drug delivery.

Thesis (Ph. D. in Biological Chemistry)--Massachusetts Institute of Technology, Dept. of Chemistry, 2013.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Major impediments to the full utility of current and potential drugs include issues of resistance and delivery. To address these challenges, in this thesis two directions of research were pursued: (1) the use of multivalent polymeric inhibitors to overcome drug resistance in human and avian influenza and (2) low-viscosity, high-concentration protein suspensions for therapeutic antibody, in particular monoclonal antibody (MAb), delivery. (1) Influenza resistance to small molecule neuraminidase (NA) inhibitors is spreading. Little emphasis, however, has been placed on alternative formulations of inhibitors. We investigated the design of multivalent antivirals, wherein small molecule ligands of viral proteins are conjugated via a linker to a linear polymeric backbone. Unexpectedly, we found that a poly-L-glutamine bearing pendant zanamivir (ZA) groups is at least as potent as those containing both ZA and sialic acid (SA). By examining the structure-activity relationship of such monofunctional conjugates, we show that the most potent one has 10% ZA attached to a neutral, high molecular weight backbone through a short alkyl linker. Importantly, we also demonstrate that such a polymer conjugate entirely compensates for weakened binding in and has 2,000-fold enhanced anti-viral potency against, ZA-resistant strains. We further evaluated this optimized inhibitor in vivo and observed that it is an effective therapeutic of established infection in ferrets and reduces viral titers up to 190-fold when used as a combined prophylactic/therapeutic in mice. Additionally, we see no evidence that the conjugate stimulates an immune response in mice upon repeat administration. (2) Typically, high doses of MAb therapeutics are required for clinical effect. Ideally, these MAbs would be delivered by subcutaneous injection of a small liquid volume. Such highly concentrated MAb solutions, however, are far more viscous than the 50 centipose (cP) permitted by the FDA. We evaluated approaches to reduce formulation viscosity by forming protein suspensions. Aqueous suspensions induced by poly(ethylene glycol), precipitating salts, or ethanol actually increased viscosity. However, non-aqueous suspensions of amorphous antibody powders in organic solvents that have s 1 hydrogen atom available for hydrogen-bonding, exhibited up to a 38-fold decrease in viscosity.
by Alisha K. Weight.
Ph.D.in Biological Chemistry

Background and aim: In situ gelling formulations are liquids that undergo a phase transition to form semi-solid gel structures within the physiological environment. Sustained release of drug products can therefore be delivered via injectable in situ gelling formulations where the gel formed in vivo acts as a drug reservoir, releasing drug via diffusion and/or degradation of the gel. Due to limitations of previously described in situ gelling formulations, the current research project aimed to address these issues and develop a biocompatible injectable in situ gelling formulation/s containing a hydrophilic polymer within a non-aqueous solvent that would gel upon contact with aqueous physiological fluid. The ideal formulation was developed to exhibit long-term stability, be able to release both hydrophilic and hydrophobic drugs over a sustained period of time without a significant initial drug burst, and be inexpensive to manufacture. Experimental: Preformulation studies were performed and then optimised to develop suitable formulations containing hydroxypropyl methylcellulose (HPMC) carboxymethyl cellulose (CMCS), or methylcellulose (MC). These lead formulations were characterised to determine suitability for injectable in situ gelling activity by assessing viscosity, syringeability and gelling behaviour, and then tested for physical stability over a 6 month period. In vitro drug release characteristics were determined for a range of hydrophilic and hydrophobic compounds before the final ideal compound, namely CMCS2a, was chosen for drug incorporation studies. A model drug, risperidone, was incorporated into the final formulation and tested for physical and chemical stability over an 8-week period.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Pharmacy
Griffith Health
Full Text

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica
In this work, novel chitosan (CHT) based microparticles were prepared using supercritical assisted atomization (SAA) and evaluated as potential carriers for sustained pulmonary drug delivery. CHT is a polysaccharide comprising of glucosamine and N-acetylglucosamine units, it is biodegradable, biocompatible and non-toxic being an interesting choice to be used as a drug carrier for inhalation therapy and belongs to the group of swellable polymers. By utilizing SAA, spherical microparticles containing a sharp particle distribution were successfully produced. Ibuprofen (IBP) and bovine serum albumin (BSA) were tested as a model small drug and as a model protein, respectively, to assess the effect on particle size and morphology when co-atomized with CHT on the SAA apparatus. The strategy developed in this work was to produce drug loaded microparticles with suitable aerodynamic characteristics that attain large geometric diameters when in contact with the lung physiological fluids by polymer swelling, reducing macrophage clearance. The microparticles produced by this method were characterized by using Morphologi G3 and Scanning electron microscopy (SEM) to assess their size distribution and morphology. To characterize the solid state properties of the particles X-ray diffraction (XRD), differential scanning calorimetry DSC and Fourier transform infrared (FTIR) were used. Porosity and surface area were determined by mercury and nitrogen porosimetry. In vitro aerosolization studies using an Andersen Cascade Impactor (ACI) were performed to determine the average emitted fraction (EF%) and the fine particle fraction (FPF). Drug-release profiles were determined by in vitro experiments at physiological pH and temperature conditions. The results obtained in this work show that SAA can be successfully used to prepare chitosan based formulations with adequate respirable fractions and sustained release of different bioactive molecules to be administered to the deep lung using dry powder inhalers (DPI).
Fundação para a Ciência e Tecnologia - contracts PEst-C/EQB/LA0006/2011, PTDC/EQU-EQU/116097/2009, Conselho de Reitores das Universidades Portuguesas (CRUP) through Luso-German Agreement A - 13/ 10 and from Fundação Calouste Gulbenkian is acknowledged

In the recent years, development of various organic and inorganic nano-sized systems has gained great interests especially for cancer diagnosis and treatment and intense researches are carried out in this area. Regarding to the recent trends for drug delivery system design, the novel approaches for drug carriers are mainly based on development of smart and nano-size drug carriers which are targeted to cancer cells. Hence, for an effective tumor-targeted delivery device, besides its chemical structure further criteria such as detection of tumor site and sensitivity to the higher temperature and lower pH of the tumor compare to rest of the body gains importance. The aim of this study is to design and prepare polysebacic anhydride (PSA) based nanocapsules (NCs) loaded with Doxorubicin (DOX) which is an anti cancer drug. In order to obtain an intelligent delivery system, drug-loaded nanocapsules were coated with pH sensitive poly (L-histidine). PSA nano-carriers were firstly loaded with DOX and then in order to introduce pH sensitivity, they were coated with poly (L-histidine). PLH-coated NCs were modified with polyethylene glycol (PEG) to prevent their macrophage uptake. Drug release profile from this system was examined in two different buffer solutions prepared as acidic (pH 4) and physiological (pH 7.4) media. The physical and chemical properties of the nano particles were characterized by Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), ultraviolet and visible absorption spectroscopy (UV-VIS), and scanning electron microscopy (SEM). In vitro studies of the prepared nanocapsules were performed on MDA-MB-231 breast cancer cells by using WST Kit 8 cell viability test. In order to obtained results, pH sensitive nanocapsules with size 230 nm exhibited cellular uptake and promising intracellular release of drug.

Recent studies have shown the numerous advantages associated with specific drug delivery to the colon, highlighting its favourable conditions and long transit time as the main advantages. A number of in vitro studies also show that the delivery of liposomes to the colon could provide further advantages due to bonding to the colonic mucosa in both healthy and inflamed regions. Despite these apparent advantages no oral liposomal formulation has been developed for targeted delivery to the colon as yet. Initially, experiments were conducted in which liposomes were directly coated with the pH responsive polymer Eudragit S100. Although the coating was shown to slow drug release in simple pH buffers, it was realised it could not protect the lipid membrane from the model bile salt sodium taurocholate. Development of the formulation moved onto the production of Eudragit S100 microspheres to provide a solid barrier to protect the liposomes. Due to the solvents required in the microsphere production it was essential to protect the liposomes, which was done by coating them with the enzyme controlled polymer chitosan. The final stage involved encapsulating chitosan-coated liposomes within the Eudragit microspheres to produce a novel, colon targeting liposome-in-microsphere (LIM) formulation.

Localized drug delivery systems have been widely studied as potential replacements for conventional chemotherapy with the capability of providing sustained and controlled drug release in specific targeted sites. They offer numerous benefits over conventional chemotherapy such as enhancing the stability of embedded drugs and preserving their anticancer activity, providing sustained and controlled drug release in the tumor site, reducing toxicity and diminishing subsequent side effects, minimizing the drug loss, averting the need for frequent administrations, and minimizing the cost of therapy. The aim of this study is to develop a localized drug delivery system with niosomes embedded in a chitosan hydrogel with targeting capabilities. The incorporation of niosomes into a chitosan hydrogel has several advantages over each individually being used. First, embedding niosomes in a chitosan hydrogel can yield control over drug release especially for small molecule drugs. Second, chitosan hydrogel may improve the release time and dosage of drugs from niosomes by protecting them with an extra barrier, resulting in tunable release rates. Third, as a localized delivery system, chitosan hydrogels can prevent the migration of niosomes away from the targeted tumor sites. Finally, chitosan has mucoadhesive property which can be used in the targeting of the tumor cells with the mucin over expression. To enhance the specific targeting, the capacity of chitosan to target MUC1 overexpression in cancer cells will be analyzed. Similarly, the incorporation of chlorotoxin in this system will be achieved and evaluated. Chlorotoxin, a 36-amino acid peptide, is purified from Leiurus quinquestriatus scorpion venom with a distinct characteristic of binding preferentially to neuroectoderma tumors such as glioma, but not to normal tissue. The overexpression of MUC1, a mucin antigen, in certain cancer cells has been used as an attractive therapeutic target in the design of a drug delivery system consisting of chitosan with a distinct mucoadhesive property. To determine the level of MUC1expression in different cell lines, Cell based Enzyme Linked Immunosorbent Assay (Cell ELISA) was developed for the first time. Attenuated Total Reflectance- Fourier Transform Infra-Red (ATR-FTIR) Spectroscopy is used to investigate the possible molecular interaction between chlorotoxin and glioma cells. This study presents a new approach in monitoring the biochemical and biophysical changes in glioma cells after being exposed to CTX. In addition to characterizing the signature spectra of CTX and glioma cells, we evaluated the differences in biochemical compositions of the spectra of the glioma cells treated with and without CTX over different incubation time periods. The results indicate that the proposed localized drug delivery system with the distinct tumor targeting features and extended release profiles would tune and control the specific delivery of chemotherapeutics in tumor sites.

Targeted therapy is a significant area of research in pharmaceutical and biomedical science. Its overall aim is to achieve maximum impact on malignant cells with minimum side effects to healthy tissue. In this thesis the capabilities of magnetic microbubbles as targeted therapeutic delivery vehicles are explored. New characterisation techniques were developed in order to understand and improve the current magnetic microbubble formulation. Electron microscopy was used to analyse the nanoscale structure of microbubble shells and observe nanoparticles attached to the shell surface. A new flow phantom was developed and the targeting of magnetic microbubbles against flow conditions corresponding to those in the human body was found to be feasible in numerous vessel sizes and flow conditions. Magnetic targeting of microbubbles was also observed in a perfused porcine liver model. Magnetic targeting was then attempted against flowing blood and a decrease in targeting efficiency observed. This was also seen for biochemical targeting and collisions with red blood cells identified as the most likely cause. Importantly, the number of magnetically targeted microbubbles significantly exceeded those targeted via biochemical interactions in both blood and water. In the second part of the thesis new types of magnetic microbubble were developed. The first exploits the fusion of nano-scale magnetic droplets with phospholipid microbubbles. In the second magnetic nanoparticles were incorporated directly into the lipid shell. The new magnetic microbubble formulation could be magnetically targeted, observed via contrast ultrasound and was successfully used to deliver siRNA to neuroblastoma cells.

The concept of chewing gum for medical purposes provides discrete, convenient administration, the potential for buccal absorption and the avoidance of first pass metabolism or gastrointestinal degradation. This work contributes to the limited information available on the release of poorly soluble drugs from medicated chewing gum formulations. Lansoprazole was chosen as a model drug due to its poor solubility and instability (under acidic conditions), thus a chewing gum formulation would be of particular benefit avoiding gastrointestinal degradation. The solubility and stability of lansoprazole in artificial saliva was found to be dependent on the pH of the solution. An increase in pH caused an increase in solubility with a significant increase between pH 9 and pH 10. At pH 6, concentrations decreased over time confirming the acid instability of lansoprazole. The use of cyclodextrins as solubilisers and stabilisers for lansoprazole were investigated; complexed lansoprazole (with Mβ-CD, 1:1) resulted in a 9 fold increase in solubility compared to free lansoprazole and remained stable at pH 6. Chewing gum formulations incorporating lansoprazole were prepared and the following excipients were investigated: Revolymer‟s® hydrophilic polymer Rev7, buffering excipients and complexed lansoprazole (with Mβ-CD, 1:1). Drug diffusion from gum surfaces was found to be limited, highlighting the need for effective mastication to ensure the timely release of the drug. In vitro release was evaluated using the EP approved masticator. Various parameters were investigated including: the type of dissolution medium, pH, chew rate and sampling and replacement volumes. Significant differences in release after 30 minutes mastication were found for gums containing Rev7 and potassium carbonate (both of which contributed to increasing the hydrophilic capacity of the gum). These gums were also softer than other formulations due to a plasticising effect on the gum base elastomer resulting in softer, less cohesive gums. Complexation was not found to have an impact on in vitro drug release from gums. The study also assessed the buccal absorption of free lansoprazole and complexed lansoprazole (with Mβ-CD, 1:1) using porcine buccal mucosae. The highest partitioning coefficient was observed for free lansoprazole at pH 6.8 due to a lower ionised fraction in combination with a lower molecular weight. Complexed lansoprazole had the highest drug flux but also had the paradoxical effect of decreasing the permeability coefficient. Overall the study contributed to increasing the understanding of factors governing the release of a poorly soluble and unstable API, lansoprazole, from a medicated chewing gum formulation. The optimised formulation would contain lansoprazole, 8 % Rev7 and potassium carbonate to provide the maximum release of drug from the gum and also facilitate buccal absorption.

Simple gel formulations may be applied to enhance the systemic and local exposure of potential compounds. The aim of this thesis is the development and characterization of controlled release formulations based on thermo-reversible poloxamer gels, which are suitable for novel drug delivery applications.  In particular co-solvents (DMSO, ethanol), mucoadhesive polymers (chitosan, alginate) and salts (sodium tripolyphosphate, CaCl2) have been used to enhance the applications of poloxamer 407 (P407) formulations in preclinical animal studies. The impact of these additives on the micellization and gelation properties of P407 aqueous solutions was studied by calorimetric methods, nuclear magnetic resonance spectroscopy (NMR) and “tube inversion” experiments. The drug release behavior of hydrophobic and hydrophilic drugs was characterized by using a membrane/membrane-free experimental setup. Finally, preliminary pharmacokinetic studies using a mouse model were conducted for screening of selected inhibitors of bacterial type III secretion and for evaluation of different formulations including P407 gel. All additives, used here, reduced the CMTs (critical micelle temperature) of dilute P407 solutions, with the exception of ethanol. The gelation temperature of concentrated P407 solutions was lowered in the presence of CaCl2, DMSO, TPP and alginate. 1H MAS (Magic Angle Spinning) NMR studies revealed that DMSO influences the hydrophobicity of the PPO segment of P407 polymers. Low concentrations of DMSO did not show any major effect on the drug release from P407 gels and may be used to improve the exposure of lead compounds in poloxamer gels. A newly developed in situ ionotropic gelation of chitosan in combination with TPP in P407 gels showed an enhanced resistance to water and reduced the release rates of model drugs. From preliminary pharmacokinetic studies in mice it was revealed that poloxamer formulations resulted in an increased plasma half-life of the lead compound.

L’étude a portée sur le développement de micro- et nanoparticules polymères destinées à l’administration orale d’insuline. Une méthode de polymérisation radicalaire a été optimisée pour formuler des micro et des nanoparticules à base d’un polymère formant des hydrogels, le poly(acide de méthacrylique). Les particules ont ensuite été modifiées par greffage de résidus cystéine pour introduire des fonctions thiol en vue de renforcer les propriétés de bioadhésion et de promoteur d’adsorption des systèmes obtenus. Les particules ont montré des propriétés intéressantes de chargement en insuline et la libération se fait selon un mode de libération pH sensible. En effet, alors que l’insuline est majoritairement retenue dans la forme pharmaceutique à pH acide correspondant à un milieu gastrique, elle est libérée à un pH neutre voire légèrement basique retrouvé au niveau de l’intestin. Les systèmes ont montré une bonne capacité à améliorer le passage de l’épithélium intestinal sur des monocouches de cellules Caco 2 et sur de l’intestin isolé monté en chambres de Ussing. Au final, ces systèmes ont permis d’induire in vivo une réduction de la glycémie chez des animaux diabétiques et après une administration orale. Les essais menés sur des insulines modifiées ont permis d’identifier une stratégie de modification intéressante basée sur l’association de l’hormone à une cyclodextrine. En revanche, nos résultats suggèrent que la PEGylation de l’insuline n’apporte aucun bénéfice
The work carried out in this thesis was aimed to develop polymer micro- and nanoparticles for the oral administration of insulin. A method of radical polymerization was optimized to design micro and nanoparticles with a hydrogel forming polymer, poly(methacrylic acid) (PMAA). The particles were further modified by the grafting of cystein residues in order to introduce thiol functions which are believed to reinforce mucoadhesive and permeation enhancing properties of the formulation. The particles showed interesting loading properties for insulin and the release of the hormone was found to be pH dependent. Although insulin was mainly retained by the hydrogel particle in releasing medium mimicking the gastric environment, the hormone was released in conditions found in the intestine. The formulated systems have shown to improve the absorption of insulin through the intestinal mucosa in in vitro models including Caco 2 cell monolayers and the Ussing chambers. The microparticles selected from the in vitro experiments for in vivo studies have shown a capacity to deliver active insulin through the oral route to diabetic rats producing a reduction of the glycemia. Tests performed with modified insulin have allowed to identify that among the two strategies followed, this consisting on the association of insulin with a cyclodextrin was the most promising while the one based on the formation of an insulin-PEG conjugate did not brought any benefice

Although liposomal accumulation at the target site is an important issue, the critical parameter defining the activity of a liposomal formulation is drug release, a factor that includes where, when, and how fast the therapeutic agent dissociates from the liposomal carrier. This point was investigated using two liposomal formulations of the anti-cancer drug mitoxantrone. Mitoxantrone was encapsulated via a pH gradient method in liposomes prepared of 1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol (Choi) (55:45 mol ratio) or 1,2 dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/Chol (55:45 mol ratio), the latter exhibiting a greater rate of drug release in vivo. Using a model of liver localized cancer consisting of BDF1 mice inoculated with either P388 or L1210 cells intravenously (/.v.), it was demonstrated that a single dose of DMPC/Chol mitoxantrone (10 mg/kg) administered i.v. resulted in 100% 60 day survival. In contrast, no long-term survivors were obtained in animals treated with free or DSPC/Chol mitoxantrone. Drug levels in the liver were determined and demonstrate that greatest drug delivery was achieved with the DSPC/Chol liposomal formulation. In an effort to address whether liposome mediated delivery or drug release is the dominant factor determining therapeutic activity, additional experiments examined the role of drug release at tumour sites where liposome accumulation is slow. As demonstrated in subcutaneous LSI80 and A431 tumours grown on the backs of SCID/RAG-2 mice, the DMPC/Chol formulation demonstrated greater activity in the LSI80 tumour model and was as efficacious as the DSPC/Chol formulation when treating A431 tumours. These data emphasize the importance of designing liposomal formulations that optimize drug biological availability rather than drug delivery. In an effort to understand factors that are important in governing the activity of DMPC/Chol liposomal mitoxantrone used to treat liver localized disease, studies modulating liposomal accumulation in the liver were completed. Two methods were used to effect reductions in liposome delivery to the liver: the use of PEG-modified lipids and hepatic mononuclear phagocyte system (MPS) blockade. Both methods reduced liposomal drug accumulation in the liver by a factor of 2 to 3 fold. A significant reduction in therapeutic activity was observed when PEG-modified lipids were incorporated into the DMPC/Chol mitoxantrone formulation; however, M P S blockade did not affect anti-tumour activity. Long term survival (>60 days) was still observed in animals where hepatic MPS blockade effected elimination o f liver Kupffer cells. It is concluded that reductions in therapy observed for the PEG-modified DMPC/Chol mitoxantrone are likely due to inhibition of cell binding and processing. Conversely it is suggested that the activity of the DMPC/Chol mitoxantrone is dependent on cell processing, but the Kupffer cells do not play a significant role in this processing event.

For controlled drug delivery applications, an ideal carrier system should release its drug payload only at the site where the therapeutic activity is required. One elegant strategy for site-selective release of drugs is to utilize the acidic sites in the body, for example, tumor sites and intracellular endocytic compartments. The objective of this thesis is to develop a series of new acid-cleavable polymeric nanoparticles for pH-triggered delivery oftherapeutics. Four new acid-cleavable benzaldehyde acetal crosslinkers have been designed and synthesized. They were then used in the generation of acid-labile polymeric nanoparticle drug carrier systems via various synthetic strategies and drug loading approaches for the delivery of therapeutics with different nature: (l) the coreshell poly(butyl acrylate)-g-poly(polyethylene glycol acrylate) nanoparticles, synthesized via the reversible addition-fragmentation chain transfer (RAFT)-mediated dispersion polymerization, were used for the delivery of hydrophobic drugs; (2) the core-crosslinked poly(hydroxyethyl acrylate)-b-poly(butyl acrylate) copolymer micelles, synthesized via the RAFT-mediated chain-extension polymerization, were used for the delivery of an antitumor drug, doxorubicin; (3) the poly(hydroxyethyl methacrylate) microgel particles, synthesized via the inverse-emulsion polymerization, were used for the delivery of biomacromolecular drugs. The designed physiochemical features such as the size, surface chemistry, cytotoxicity and the pH-triggered drug release properties of the developed carrier systems have been assessed. The synthesized systems offered release of the drug payload at slightly acidic conditions. The structural integrity of the polymeric carriers remained intact in the physiological, neutral pH conditions. The results support the potential value of the developed systems to be used for acidic-site delivery of therapeutics e.g. tumor sites and intracellular compartments. The content of this thesis has been published as three peer-reviewed international journal articles.

Background and purpose: Biodegradable polymers, such as PLGA, are delivery vehicles used to treat posterior segment eye disease, but suffer from poor drug loading and initial burst release. This thesis describes a ‘system-within-system’, PLGA microparticles incorporating chitosan-based nanoparticles, for ranibizumab delivery, and chitosan-containing micelles for transscleral rapamycin delivery. Methods: Synthesis of chitosan-N-acetyl-L-cysteine (CNAC) and octanoyl-chitosan-poly (ethylene glycol) (OChiPEG) were confirmed using spectroscopy. Chitosan/TPP, chitosan/TPP-HA, CNAC and CNAC/TPP nanoparticles containing ranibizumab were prepared then incorporated in PLGA microparticles and characterised for their size, zeta potential, morphologies, solid-state properties, morphology, protein loading, stability, release, in vivo antiangiogenic activity and effects on cell viability. Rapamycin-loaded micelles comprised of OChiPEG and chitosan-incorporated TPGS, CPEG and TPGS/CPEG were also prepared. Micelles were analysed for their size, zeta potential, morphology, stability, solid-state properties, rapamycin entrapment efficiency and ex vivo scleral retention and permeation. Results: Chitosan-based ranibizumab-loaded nanoparticles measured 17 – 350 nm with a zeta potential of -1.4 to + 12 mV; microparticles measured 3.0 – 6.6 µm (-12 to + 9.7 mV). PLGA microparticles appeared mostly spherical; those prepared with chit/TPP, CNAC and CNAC/TPP had spherical nanoparticles on their surface. Microparticle protein content ranged from 13 – 69%. All preparations showed burst release except for the CNAC containing microparticles, which had the slowest release. Ranibizumab released from PLGA microparticles maintained structural integrity, and activity in cell culture. Chit/TPP-HA nanoparticles containing ranibizumab showed enhanced antiangiogenic activity relative to native ranibizumab. Microparticles showed no effect on cell viability up to a concentration of 12.5 mg/mL. Rapamycin loaded micelles measured 11 – 41 nm, with a zeta potential of -1.2 to + 6.8 mV, entrapment efficiency of 75 - 97% and a scleral retention of 7.5 – 44 µg/g. Conclusion: Micelles showed enhanced scleral retention with potential for topical ocular delivery of poorly soluble drugs. The CNAC-containing preparation has potential for the intraocular delivery of protein-based drugs.

Over recent years the delivery of nanosized drug particles has shown potential in improving bioavailability. Drug nanosizing is achieved by 'top-down' and by 'bottom-up' approaches. Owing to limitations associated with the top-down techniques, such as high energy input, electrostatic effects, broad particle size distributions and contamination issues, great interest has been directed to alternative bottom up technologies. In this study, the hypothesis that microreactors can be used as a simple and cost-effective technique to generate organic nanosized products is tested using three steroids (hydrocortisone, prednisolone and budesonide). Arrested antisolvent nanoprecipitation using ethanol (solvent) and water (antisolvent) was conducted within the microreactors. To enable experimental design for the microreactor studies, solubility profiles in different ethanol-water combinations at 25 °C were explored. All three drugs' solubility increased with increasing ethanol concentration showing maxima at 80-90 % v/v ethanol-water mixtures. Because of the complex multivariate microfluidic process, artificial neural network modelling was then employed to identify the dominant relationships between the variables affecting nanoprecipitation (as inputs) and the drug particle size (as output). The antisolvent flow rate was found to have the major role in directing drug particle size. Based on these successful findings, the potential of preparing pharmaceutical nanosuspensions using microfluidic reactors was researched. A hydrocortisone (HC) nanosuspension (NS) was prepared by introducing the generated drug particles into an aqueous solution of stabilizers stirred at high speed with a propeller mixer. A tangential flow filtration system was then used to concentrate the prepared NS. Results showed that a stable narrow sized HC NS of amorphous spherical particles 500 ± 64 nm diameter and zeta potential -18 ± 2.84 mV could be produced. The ocular bioavailability of a microfluidic precipitated HC NS (300 nm) was assessed and compared to a similar sized, milled HC NS and HC solution as a control. The precipitated and the milled NS achieved comparable AUC0-9h of 28.06 ± 4.08 and 30.95 ± 2.2, respectively, significantly (P < 0.01) higher than HC solution (15.86 ± 2.7). These results illustrate the opportunity to design sustained release ophthalmic formulations. Going nano via microfluidic precipitation was also exploited to tailor budesonide (BD) NS for pulmonary administration. The in vitro aerosolization by nebulization of a BD NS was studied in comparison with a commercial BD microsuspension. Overall, the fine particle fraction generated from BD NS (56.88 ± 3.37) was significantly (P < 0.05) higher than the marketed BD (38.04 ± 7.81). The mean mass aerodynamic diameter of BD NS aerosol (3.9 ± 0.48 μm) was significantly smaller (P < 0.05) than the microsuspension (6.2 ± 1.09 μm) indicating improved performance for BD NS. In conclusion, findings of this study support the hypothesis of using microfluidic nanoprecipitation as a promising and economical technique of drug nanosizing.

Over recent years the delivery of nanosized drug particles has shown potential in improving bioavailability. Drug nanosizing is achieved by ¿top-down¿ and by ¿bottom-up¿ approaches. Owing to limitations associated with the top-down techniques, such as high energy input, electrostatic effects, broad particle size distributions and contamination issues, great interest has been directed to alternative bottom up technologies. In this study, the hypothesis that microreactors can be used as a simple and cost-effective technique to generate organic nanosized products is tested using three steroids (hydrocortisone, prednisolone and budesonide). Arrested antisolvent nanoprecipitation using ethanol (solvent) and water (antisolvent) was conducted within the microreactors. To enable experimental design for the microreactor studies, solubility profiles in different ethanol-water combinations at 25 °C were explored. All three drugs¿ solubility increased with increasing ethanol concentration showing maxima at 80-90 % v/v ethanol-water mixtures. Because of the complex multivariate microfluidic process, artificial neural network modelling was then employed to identify the dominant relationships between the variables affecting nanoprecipitation (as inputs) and the drug particle size (as output). The antisolvent flow rate was found to have the major role in directing drug particle size. Based on these successful findings, the potential of preparing pharmaceutical nanosuspensions using microfluidic reactors was researched. A hydrocortisone (HC) nanosuspension (NS) was prepared by introducing the generated drug particles into an aqueous solution of stabilizers stirred at high speed with a propeller mixer. A tangential flow filtration system was then used to concentrate the prepared NS. Results showed that a stable narrow sized HC NS of amorphous spherical particles 500 ± 64 nm diameter and zeta potential ¿18 ± 2.84 mV could be produced. The ocular bioavailability of a microfluidic precipitated HC NS (300 nm) was assessed and compared to a similar sized, milled HC NS and HC solution as a control. The precipitated and the milled NS achieved comparable AUC0-9h of 28.06 ± 4.08 and 30.95 ± 2.2, respectively, significantly (P < 0.01) higher than HC solution (15.86 ± 2.7). These results illustrate the opportunity to design sustained release ophthalmic formulations. Going nano via microfluidic precipitation was also exploited to tailor budesonide (BD) NS for pulmonary administration. The in vitro aerosolization by nebulization of a BD NS was studied in comparison with a commercial BD microsuspension. Overall, the fine particle fraction generated from BD NS (56.88 ± 3.37) was significantly (P < 0.05) higher than the marketed BD (38.04 ± 7.81). The mean mass aerodynamic diameter of BD NS aerosol (3.9 ± 0.48 ¿m) was significantly smaller (P < 0.05) than the microsuspension (6.2 ± 1.09 ¿m) indicating improved performance for BD NS. In conclusion, findings of this study support the hypothesis of using microfluidic nanoprecipitation as a promising and economical technique of drug nanosizing.
Egyptian Government (Ministry of High Education)

In this thesis, the capability of the electrohydrodynamic atomization (EHDA) process for preparing drug delivery carriers consisting of biodegradable polymeric particles with different sizes and shapes was explored. The first part of the thesis describes a detailed investigation of how the size, morphology and shape of the particles generated can be controlled through the operating parameters; specifically the flow rate, applied voltage and the properties of the solutions. Diameter and shape of the particles were greatly influenced by viscosity and applied voltage. The mean size of the particles changed from 340 nm to 4.4 μm as the viscosity increased from 2.5 mPa s to 11 mPa s. Also, using more concentrated polymer solution (30 wt%) and higher applied voltage (above 14 kV) were found to be ideal for promoting chain entanglement and shape transition from spherical to oblong to a more needle-like shape. Estradiol-loaded micro and nanoparticles were produced with mean sizes ranging from 100 nm to 4.5 μm with an encapsulation efficiency ranging between 65% to 75%. The in vitro drug release profiles of the particles started with an initial short burst phase and followed by a longer period characterised by a lower release rate. Two strategies were developed to tailor these profiles. First, ultrasound was explored as a non-invasive method to stimulate “on demand” drug release from carrier particles. Systematic investigations were carried out to determine the effect of various ultrasound exposure parameters on the release rate in particular output power, duty cycle and exposure time. These three exposure parameters were seen to have a significant enhancing effect upon the drug release rate (up to 14%). The second strategy explored was coating the surface of the particles with chitosan and gelatin. This enabled control and reduction of the prominence ‘burst release’ phase without affecting other parts of the release profile. Coating the particle surface with 1 wt% chitosan solution considerably reduces the initial release by 62%, 60% and 42% for PLGA 2 wt%, 5 wt% and 10 wt%, respectivly in the first 72 hours This work demonstrates a powerful method of generating micro and nano drug-loaded polymeric particles, with modified release behaviour and with control over the initial release.

Synthesis and characterization studies of promising nano-sized hydrogel composites based on nisopropylacrylamide and magnetite have been studied. N-isopropylacrylamide (NIPA) gel component was used as a carrier of various drugs, magnetite was used as a magneto-responsive component. Presence of magnetite it was proved by EPR method. Composite nanoparticles were characterized by electron microscopy (TEM) and by dynamyc light scattering (DLS) method. It was shown that the average size of nanoparticles is 50 or 100 nm, depending on the method of preparation. The hydrogel is characterized by clear phase transition between swollen and collapsed state upon heating above 32⁰C. Rapid release of the incorporated drug (as a model was used the photosensibilizer -Methylene Blue) observed during thermoresponsive nanocomposite gels heating in the physiologically acceptable range, but still above phase transition temperature (up to 40–50 ⁰C), allows application of the discussed drug delivery systems in medical hyperthermia.

La industria farmacéutica hoy en día tiene que afrontar varios retos ya que el 40\% de los compuestos resultantes de los programas de selección combinatorios son insolubles en agua. Como consecuencia, estas moléculas presentan dificultades a la hora de ser procesadas. Una de las estrategias más implementadas para aumentar la velocidad de disolución de estos nuevos fármacos es su formulación como microparticulas. Las propiedades de los ingredientes activos están directamente relacionadas con las características de las partículas tales como el tamaño, la forma, la estructura cristalina y la morfología. Concretamente, el control del tamaño y la forma de un fármaco son de vital importancia ya que estos dos parámetros influencian gran cantidad de propiedades físicas, sus posibilidades de procesado y calidad. Además, las compañías farmacéuticas tienen la necesidad urgente de desarrollar procesos de bajo impacto medioambiental, en particular, para reducir el empleo de disolventes orgánicos volátiles en los procesos de producción de fármacos así como el nivel de residuos en el producto acabado. Las estrictas limitaciones que sufre hoy en día la industria farmacéutica para obtener productos de alta calidad junto con la creciente preocupación de la sociedad por la seguridad y el medioambiente hacen que la implementación de técnicas más eficientes y más respetuosas con el medioambiente sea en una necesidad urgente. Los fluidos comprimidos (CF) surgieron en los años 80s y presentan unas propiedades únicas para la preparación de principios activos con un control excepcional de las variables de operación que permiten modelar las propiedades finales de los fármacos de una manera sostenible, como se detalla en el Capitulo 1 de esta Tesis. Una de las aplicaciones más exitosa de los fluidos comprimidos es en la producción de fármacos. En las cristalizaciones con disolventes convencionales, los tamaños de partícula deseados se obtienen sometiendo a los fármacos a procesos de molienda. En los procesos a partir de fluidos comprimidos, la formación de partículas se hace de manera controlada para obtener las propiedades finales deseadas en una sola etapa. Esto significa, que una vez que la partícula se forma, no tiene que someterse a tensiones térmicas ni mecánicas. Esta característica hace que las técnicas basadas en el uso de fluidos comprimidos sean adecuadas para producir biomoléculas. Además, los procesos con fluidos comprimidos presentan un gran potencial para su aplicación a gran escala. En base a la necesidad de implementar procesos sostenibles para la producción de principios activos con un tamaño y forma definidos, el objetivo de esta Tesis es expandir la bondad de los procesos de precipitación basados en fluidos comprimidos. Concretamente, el Capitulo 2 está centrado el uso del DELOS, un proceso basado en CFs, para preparar microparticulas cristalinas de activos con poca solubilidad en agua y que presentan problemas a la hora de ser procesados por técnicas convencionales. Otra prometedora estrategia para formular compuestos insolubles en agua es su formulación como suspensiones acuosas donde el fármaco se encuentra suspendido en forma de partícula micrónica en un medio acuoso. En este contexto, el Capitulo 3 explora la aplicación del método de una sola etapa DELOS-susp para la obtención de suspensiones acuosas de fármacos insolubles en agua de tamaño micrónico. Por último, con el objetivo de expandir el uso de los fluidos comprimidos, la parte final de esta Tesis ha estado dedicada a la investigación y caracterización a nivel molecular de sistemas tipo microemulsión sin surfactantes formados en mezclas "agua/disolvente orgánico/CO2" a alta presión. Estos líquidos nanoestructurados se pueden considerar como prometedores disolventes respetuosos con el medioambiente y como plantillas para la preparación de materiales nanoparticulados.
The pharmaceutical industry nowadays is facing several challenges, as more than 40 % of compounds identified through combinatorial screening programs are poorly soluble in water. These molecules are difficult to formulate using conventional approaches and are associated with innumerable formulation-related performance issues. Formulating these compounds as pure drug micro particles is one of the newer drug-delivery strategies applied to this class of molecules. The bioperformance of drugs depends on specific characteristics of particles such as size, surface, crystal structure and morphology. Concretely, the control of particle size and shape is of vital relevance as they influence a large variety of important physical properties, manufacturing processability and quality attributes. Moreover, pharmaceutical companies are more and more urged to develop production processes with very low environmental impact in particular for reducing the use of volatile organic compounds in medicine manufacturing as well as the residues in the finished product. In the case of pharmaceutical industry, requirements for high-quality products and society concerns about health and environments make the implementation of new efficient and environmentally respectful technologies for the preparation of drugs with tailored properties an urgent necessity. Compressed fluids (CF), which emerge in the early 80's, present unique properties for the eco-efficient production of Active Pharmaceutical Ingredients (APIs) with an exceptional control of the operational variables that allows tuning the final properties of the active compounds, as detailed in Chapter 1 of this Thesis. Among the most successful applications of CFs, particle engineering of pharmaceutical actives seems to be at the moment, the area with the highest blooming. In contrast to conventional particle formation methods, where a larger particle is originally formed and then milled to the desired size, CF technology involves growing particles in a tailored manner to reach the desired final physical properties. This means that the solid particle, once formed, does not have to undergo any thermal nor mechanical stresses, as happens in conventional techniques. This feature makes CF technology amenable to produce biomolecules and other sensitive compounds in their native pure state. In addition, CF-based technologies also present an enormous potential for large scale processing. In light of the need of implementing environmentally friendly processes for the production of APIs with controlled size and shape, this Thesis has been devoted to expand the goodness of CF-based methodologies. Concretely, Chapter 2 focuses on the use of DELOS, a CF-based precipitation process, to prepare micronized crystalline particles of poorly soluble actives with low bioavailability and problematic processing by conventional techniques. Another promising approach to increase the bioavailability of poor soluble drugs is their formulation as micro particles suspended in an aqueous media forming aqueous suspensions. In this context, Chapter 3 explores the application of DELOS-susp as a new one-step method for preparing aqueous suspensions of micronized actives. Finally, with the objective of expanding the use of CF-based process, the last part of this Thesis endeavors to investigate and characterize the organization, at the molecular level, of surfactant-free microemulsion-like systems formed in "water/organic solvent/\CO2" pressurized systems. These nanostructured liquids can be regarded as universal green solvents and could be used as nano templates.

Les sphéroïdes multicellulaires tumoraux (SMT) constituent un outil prometteur dans le domaine de l’étude biologique des tumeurs. Le but de la thèse était de développer une technique de la formation de SMT et de démontrer la disponibilité de ces sphéroïdes comme modèle in vitro 3D pour tester l’efficacité de principes actifs anticancéreux ainsi que celle de formulations de délivrance de médicaments. L'effet d’auto-assemblage de cellules induit par une addition des peptides RGD cycliques a été étudié pour 16 lignées cellulaires de différentes origines. Le peptide cyclique RGDfK et sa modification avec le cation triphenylphosphonium (TPP) ont permis de mettre en évidence l’induction de formation de sphéroïdes. Les sphéroïdes ont été employés comme modèles pour évaluer la cytotoxicité de principes actifs antitumoraux (doxorubicine, curcumine, temozolomide) et un certain nombre de formulations nano- et micrométriques (microréservoirs, nano-émulsions et micelles)
Multicellular tumor spheroids (MTS) are a promising tool in tumor biology. The aim of the Thesis was to develop a novel highly reproducible technique for MTS formation, and to demonstrate the availability of these spheroids as 3D in vitro model to test anticancer drugs and drug delivery vehicles. Cell self-assembly effect induced by an addition of cyclic RGD-peptides directly to monolayer cultures was studied for 16 cell lines of various origin. Cyclo-RGDfK peptide and its modification with triphenylphosphonium cation (TPP) were found to induce spheroid formation. The spheroids were used as a model to evaluate the cytotoxicity of antitumor drugs (doxorubicin, curcumin, temozolomide) and a number of nano- and micro- formulations (microcontainers, nano-emulsions and micelles)

This thesis describes the preparation and characterization of core-shell noble metal-magnetite hybrid hollow nanocomposites utilizing hierarchical architecture. The hollow magnetite (hFe3O4) nanoparticles were prepared by hydrothermal method, forming the cavity via Oswald ripening. Further surface modifications involved both inorganic and organic coatings, conferring the intracellular drug delivery ability and the catalytic enhancement. In the first part, a series of hierarchical core-shell nanostructures flower-like hFe3O4@AlOOH was synthesized through solvothermal method and sol-gel process. The formation of cavity accessible hFe3O4@γ-AlOOH was achieved using silica-templated solvothermal treatment where the Kirkendall effect was observed. The morphologies of the as-prepared nanocomposites were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), dynamic light scattering (DLS), thermogravimetric analysis (TGA) and Fourier-transform infrared spectroscopy (FTIR). Then, the nano-encapsulation of platinum drug using hollow magnetite and its derivatives, has been developed with improved loading efficiency via co-solvent system. A dimethylformamide/water co-solvent system was found to be the most efficient system to encapsulate water-insoluble cisplatin. The platinum content was further quantitatively and qualitatively analyzed by inductively coupled plasma mass spectrometry (ICP-MS) and FTIR spectroscopy. The enhancement of loading efficiency could be driven by emulsification due to the diffusion of hydrophobic cisplatin into the hollow cavity of iron oxide nanoparticles. By incorporating water, the loading efficiency of hFe3O4 and hFe3O4@γ-AlOOH increased from 1-2% to 27% and from 6% to 54%, respectively. The grafting of cisplatin on AlOOH nanoflakes might account for the high loading efficiency of flower-like hFe3O4@AlOOH. As a complement to naked hFe3O4, a cell-penetrating poly(disulfide)s (CPD)-decorated hollow iron oxide nanoparticle was synthesized by immobilizing both cysteine and MPTMS as an initiator, followed by in situ polymerization to form hFe3O4-Cys-CPD-CONH2 and hFe3O4-MPS-CPD-CONH2. The morphologies were characterized by TEM/energy-dispersive X-ray spectroscopy (TEM/EDX) and the compositions of the as-prepared iron oxide nanocomposites were characterized by TGA, FTIR and X-ray photoelectron spectroscopy (XPS) and ICP-MS. The CPD coating not only serve as a protective layer, but also prevent the encapsulated cisplatin from a premature release. The hFe3O4-MPS-CPD-CONH2 exhibit promising features for the intracellular delivery of cisplatin, demonstrating a glutathione (GSH)-responsive drug release. Comparing with other hFe3O4 nanoparticles, an enhancement of cellular uptake of hFe3O4-MPS-CPD-CONH2 could be observed by optical microscope, showing rapid accumulation of the hFe3O4-MPS-CPD-CONH2 nanocomposites in the primary human renal proximal tubular epithelial cells (HRPTEpiCs) cell in 2 h. At 24 h, hFe3O4 (F), hFe3O4-MPS (FS) and hFe3O4-MPS-CPD-CONH2 (FSC) together with cisplatin treatment did not cause any significant cytotoxicity to the cells when the particle concentration is less than 10 µg/mL. Interestingly, FSCC showed a certain extent of toxicity with increasing Fe and Pt concentration along with the treated time. It may suggest that the hFe3O4-MPS-CPD-CONH2 nanoparticle, as a cisplatin carrier, could enhance the drug efficiency by increasing cellular uptake of the nanoparticles in HRPTEpiCs together with the boosted cytotoxicity. Based on these data, cisplatin- hFe3O4-MPS-CPD-CONH2 (FSCC) treatments with the concentration less than 20 µg/mL and duration no more than 24 h could maintain around 70% of the cell viability of the HRPTEpiCs. The hypothesis, at which CPD serves as an efficient carrier for intracellular cisplatin delivery, could be confirmed by both microscopic images and the cell viability test. In the second part, a series of Au/Fe3O4 hybrid nanocomposites was prepared to investigate their catalytic efficiencies using 4-nitrophenol reduction as a model system. The flower-like hFe3O4@γ-AlOOH@SiO2-NH2@Au was prepared by using protonated ammonium on hFe3O4@γ-AlOOH@SiO2-NH2 to entangle gold nanoparticles (AuNPs) via electrostatic attraction. In comparison to numerous of catalytic studies, the turnover frequency (TOF) of hFe3O4@γ-AlOOH@SiO2-NH2@Au shows a superior conversion rate up to 7.57 min-1 (4-nitrophenol per Au per min) for the 4-nitrophenol using sodium borohydride as a reductant. A rapid conversion of 4-nitrohpenol was observed using flower like composites that converted the 4-nitrophenol within 2 min. Our result suggests that silica residue hinders the reduction rate of the 4-nitrophenol. A significant deviation from pseudo first order was observed for densely AuNPs-functionalized nanoflower system, hFe3O4@γ-AlOOH@SiO2-NH2@Au2X, which is different from most of the 4-nitrophenol reductions reported in literature. The hFe3O4@γ-AlOOH@SiO2-NH2@Au also demonstrates catalytic activity when heated up to 800 °C before reduction. The recyclability was examined using magnetically recycled hFe3O4@γ-AlOOH@SiO2-NH2@Au, which showed insignificant decrease in the catalytic efficiency. To prove the concept, platinum nanoparticles (PtNPs) immobilized hFe3O4@γ-AlOOH@SiO2-NH2@Pt and hFe3O4@γ-AlOOH@SiO2-NH2@Pt/Au were also prepared via electrostatic attraction to verify the feasibility of endowing modular functionality via post modification.

Dry powder inhalers (DPIs) are advantageous for delivering medication to the lungs for the treatment of respiratory diseases because of the stability of the powders, relative low cost, synchronization of inhalation and dose delivery, and many design options that can be used for optimization. However, currently marketed DPIs are very inefficient in delivering medications to the lungs. This study has developed multiple new high efficiency DPIs for use with spray dried excipient enhanced growth (EEG) powder formulations based on the following platforms: capsule-based for oral inhalation, high-dose for oral inhalation, inline with 3D rod array dispersion, and inline with capillary jet dispersion. The capsule-based DPIs for oral inhalation implemented a 3D rod array for aerosol dispersion with optimal designs producing mass median aerodynamic diameters (MMADs) in the range of 1.3-1.5 µm and emitted doses in the range of 79-81%. Keys to inhaler success were the orientation of the capsule and inclusion of the 3D rod array. For the high-dose oral inhaler, performance was similar to the optimized capsule-based devices, while aerosolizing a much larger mass of powder. Surprisingly, removal of the fluidized bed of spheres improved performance producing a simple high dose device containing only a single dose sphere. The inline device using the 3D rod array was effective in producing particles of approximately 1.5 µm, at flow rates consistent with high flow therapy using a 1 L ventilation bag as the delivery mechanism. Using a capillary jet as the dispersion mechanism, further advances were made to allow for both delivery using a low volume (LV) of air and delivery in low flow therapy. This easily adaptable platform was able to produce a high quality aerosol out of a nasal cannula with an ED greater than 60% and a size (~2 µm) that should produce minimal extrathoracic losses. In conclusion, this study demonstrates (i) the design and optimization of DPIs capable of delivering EEG aerosols to the lungs using oral inhalation, (ii) the ability to deliver EEG aerosols using N2L aerosol administration, and (iii) the design of a new flexible LV-DPI device that is easily adaptable to multiple patients and delivery platforms, which are greatly needed in clinical environments.

The results from this thesis leads to the following conclusions; HUM is a useful tool that fills a gap in the in vitro methods previously available to study nasal drug delivery. Using HUM, the pig respiratory nasal mucosa can obtain acceptable viability and retain it longer than the period of time needed for a transport experiment. HUM has proven to be an appropriate tool for the study of liquids in low volume, gels, both unmodified and with controlled release properties, and particle suspensions. The potential local toxicity of formulations such as controlled release gels and surfactants could be evaluated and graded using HUM. The estimation of the apparent permeability can be corrected on a mathematical basis, for substances that bind to the chamber material. As seen using HUM, unmodified gels from Carbopol 934 (C934) are well tolerated by the nasal mucosa and may consequently be suitable for nasal administration. The release rate of testostenone, dihydroalprenolol and hydrocortisone from C934 gels can be successfully sustained. Protein-conjugated starch microparticles, intended to function as a vaccine carrier system, were taken up by non-ciliated epithelial cells of the pig respiratory nasal mucosa after incubation using HUM. The concentration-dependent effects on permeability and transepithelial electrical resistance on Caco-2 cells, of a series of nonionic polyoxyethylene surfactants, correlated with surfactant structure. Similar effects were seen on pig nasal mucosa using HUM, but the nasal mucosa appeared to be more tolerant to the surfactants than the intestinal cell model.

The nasal route has advantages for several classes of drugs e.g. involved in migrain treatment, nicotine substitution therapy and mucosal vaccination. The increased development of a variety of substances, in a variety of formulation types, has increased the demand for suitable investigational tools. It is in this context that the horizontal Ussing chamber method (HUM) was developed. Using HUM, the studied formulation can be applied on the mucosa without additional buffer, giving an in vivo-like situation and the possibility to study solid and semi-solid formulations. Furthermore, the influence of gravity will not result in uneven distribution of the formulation.

ABSTRACT

This project aimed at developing a drug delivery system (DDS) able to enhance the stability and

residence time in vivo of antibodies (Abs). The system will deliver drug by the subcutaneous

route (SC), while ensuring accurate control of the drug release and the resulting plasmatic level. This technology platform will allow to reduce frequency of injection, potentially decrease side effects and maintain high concentration of Abs which will improve life of patient having chronic disease such as autoimmune and inflammatory disease. Biodegradable synthetic polymer-based formulations (polylactide-co-glycolide (PLGA)) were selected as carriers for encapsulated Abs. This was because they offer good protection for the Abs and allow sustained release of the Abs for a controlled period of time. After the evaluation of different encapsulation methods such as the water-oil-in-water (w/o/w) and the solid-in-oil-inwater

(s/o/w) processes, the encapsulation of the Ab in solid state (s/o/w) appeared to be more appropriate for producing Ab-loaded PLGA microspheres (MS). It allowed us to maintain the

Ab in a monomeric conformation and to avoid the formation of unsoluble aggregates mainly present at the water/oil interface. The first part of the project was the optimization of both the method for producing the Ab solid particles (spray-drying process) and the encapsulation of these Ab solid particles into the polymeric MS (s/o/w process) by design of experiment (DoE). These optimizations were carried out using a bovine polyclonal immunoglobulin G (IgG) as model molecule. In further optimization of the spray-drying process by (DoE), aqueous Ab solutions were spray-dried using a mini Spray-Dryer assembly with a 0.7 mm spray nozzle. In accordance with the particle size (d(0.5) ~5 μm), the stability (no loss of monomer measured by

size exclusion chromatography (SEC) and the yield of the spray-drying process (> 60 % w/w), the process parameters were set of follow: 3 mL/min as liquid feed flow rate, 130°C /75°C as inlet temperature (inlet T°) / outlet temperature (outlet T°), 800 L/h as atomization flow rate and

30 m3/h as drying air flow rate. For the s/o/w, the methylene chloride (MC) commonly used for

an encapsulation process was replaced by ethyl acetate (EtAc), which was considered as a more

suitable organic solvent in terms of both environmental and human safety. The effects of several processes and formulation factors were evaluated on IgG:PLGA MS properties such as: particle size distribution, drug loading, IgG stability, and encapsulation efficiency (EE%). Several formulations and processing parameters were also statistically identified as critical to get reproducible process (e.g. the PLGA concentration, the volume of the external phase, the emulsification rate, and the quantity of IgG microparticles). The optimized encapsulation

method of the IgG has shown a drug loading of up to 6 % (w/w) and an encapsulation efficiency

of up to 60 % (w/w) while preserving the integrity of the encapsulated antibody. The produced MS were characterized by a d(0.9) lower than 110 μm and showed burst effect lower than 50 %(w/w). In the second part of the project, the optimized spray-drying and s/o/w processes

developed with the IgG were applied to a humanized anti-tumor necrosis factor (TNF) alpha

MAb to confirm the preservation of the MAb activity during these processes. The selected s/o/w method allowed us to produce MAb-loaded PLGA MS with an appropriate release profile up to 6 weeks and MAb stability. In order to maintain the Abs’ activity, both during encapsulation and

dissolution, the addition of a stabilizer such as trehalose appeared to be crucial, as did the

selection of the PLGA. It was demonstrated that the use of a PLGA characterized by a 75:25

lactide:glycolide (e.g. Resomer ® RG755S) ratio decreased the formation of low molecular weight species during dissolution, which led to preserve Abs activity through its release from the

delivery system. Furthermore, the release profile was adjusted according to the type of polymer

and its concentration. E.g. 10 % w/v RG755S allowed Ab MS with a release time of 6 weeks to

be obtained. The optimization of both the formulation and the encapsulation process allowed

maximum 13 % w/w Ab-loaded MS to be produced. It was demonstrated that the Ab-loaded PLGA MS were stable when stored at 5°C for up to 12 weeks and that the selection of the appropriate type of PLGA was critical to assuring the stability of the system. The better stability observed when using a PLGA characterized by a 75:25 lactide:glycolide ratio was attributed to

its slower degradation rate. Finally, the sustained release of Ab from the developed MS and the preservation of its activity was confirmed in vivo in a pharmacokinetic (pK) study realized in

rats. In conclusion, the application of the concept of entrapment into a polymer matrix for

stabilization and sustained release of biological compounds was demonstrated through this work.

RÉSUMÉ

Ce projet a pour but de développer un système de délivrance de médicament capable d’augmenter la stabilité et le temps de résidence in vivo des anticorps. Ce système sera administré par voie sous-cutanée et permettra un control précis de la libération du produit et de son niveau plasmatique. Cette plateforme technologique nous permettra de réduire la fréquence d’injection, de réduire potentiellement les effets secondaires et de maintenir des concentrations élevées en anticorps tout en améliorant la vie des patients atteints de maladies chroniques autoimmunes ou inflammatoires. Les formulations à base de polymères synthétiques, biodégradables (PLGA) ont été sélectionnés comme véhicules pour encapsuler les anticorps. Ils offrent en effet une bonne protection pour les anticorps and permettent une libération contrôlée de ceux-ci pendant une période définie. Après l’évaluation de différents méthodes d’encapsulation tels que les procédés d’eau-dans-huile-dans-eau (w/o/w) et solide-dans-huile-dans-eau (s/o/w), l’encapsulation des anticorps sous forme solide apparaissait plus apporpriée pour produire des microsphères de polymère chargées en anticorps. Cette technique nous permettait de maintenir l’anticorps sous sa forme monomérique et d’éviter la formation d’agrégats insolubles qui apparaissaient principalement à l’interface eau/huile. La première partie du projet a été d’optimiser à la fois la méthode nous permettant d’obtenir les anticorps sous forme de particules solides (spray-drying) et la méthode d’encapsulation de ces particules d’anticorps dans les microsphères de polymères. Cela a été réalisé par des plans d’expérience en utilisant une IgG bovine polyclonale comme molécule modèle. Durant l’optimisation du procédé de spray-drying,

les solutions aqueuses d’anticorps ont été atomisées en utilisant le mini Spray-Dryer assemblé avec une buse de pulvérisation d’un diamètre de 0.7 mm. En accord avec la taille particulaire (d(0.5) ~5 μm), la stabilité (absence de perte en monomère mesurée par chromatographie d’exclusion de taille et le rendement d’atomisation (> 60 % w/w), les paramètres d’atomisation ont été fixés: 3 mL/min pour le débit de liquide, 130°C /75°C pour la température d’entrée / température de sortie, 800 L/h pour le débit d’air d’atomisation et 30 m3/h pour le débit d’air de séchage. Pour le s/o/w, le dichlorométhane communément utilisé dans les procédés d’encapsulation a été remplacé par l’acétate d’éthyle qui est considéré comme un meilleure solvant organique en terme d’environnement et de sécurité. Les effets de plusieurs paramètres de fabrication ou de formulation ont été évalués sur les propriétés des microsphères polymériques d’anticorps (distribution de taille particulaire, taux de charge en anticorps, stabilité de l’anticorps et efficacité d’encapsulation). Plusieurs paramètres de fabrication et de formulation ont été statistiquement identifiés comme critiques pour obtenir un procédé reproductible (par exemple. La concentration en PLGA, le volume de phase externe, la vitesse d’émulsification et la quantité d’anticorps). La méthode d’encapsulation ainsi optimisée permettait d’obtenir un taux

de charge jusqu’à 6% (w/w) avec une efficacité d’encapsulation jusqu’à 60 % (w/w) tout en

préservant l’intégrité de l’anticorps encapsulé. Les microsphères produites étaient caractérisées

par un d(0.9) inférieur à 110 μm et montraient une libération après 24 h inférieure à 50 % (w/w).

Dans le seconde partie du projet, les procédés d’atomisation et d’encapsulation développés avec

l’IgG ont été appliqués à un anticorps monoclonal anti-TNF alpha humanisé pour confirmer la

conservation de l’activité de l’anticorps pendant ces procédés. La méthode s/o/w sélectionnée

permettait de produire des microsphères de PLGA chargées en anticorps avec un profil de libération jusqu’à 6 semaines et un maintien de la stabilité de l’actif. Afin de maintenir l’activité de l’anticorps, à la fois pendant le procédé d’encapsulation et pendant la libération, l’ajout d’un stabilisant tel que le tréhalose est apparu crucial ainsi que le choix du type de PLGA. Il a été démontré que l’utilisation du PLGA caractérisé par un ratio lactide :glycolide de 75 :25 (par exemple, Resomer ® RG755S) diminuait la formation d’espèces de faible poids moléculaire

pendant la dissolution. Cela contribuait à préserver l’activité de l’anticorps durant la libération à partir des microsphères. De plus, le profil de libération était modulé en fonction du type de polymère et de sa concentration. Par exemple, l’utilisation d’une solution à 10 % w/v RG755S conduisait à la production de microsphères d’anticorps avec un temps de libération sur 6

semaines. L’optimisation de la formulation et du procédé d’encapsulation a permis de produire

des microsphères avec des taux de charge en anticorps de maximum 13 % w/w. Il a été démontré

que ces microsphères, stockées à 5°C, étaient stables jusqu’à 12 semaines et que la sélection du

type de PLGA était critique pour assurer la stabilité du système. La meilleure stabilité a été

obtenue en utilisant le PLGA caractérisé par un ratio lactide :glycolide de 75 :25. Cela a été

attribué à sa plus faible vitesse de dégradation. Enfin, la libération contrôlée de l’anticorps à

partir de ces microsphères et la conservation de son activité ont été confirmées in vivo lors d’une

étude pharmacocinétique réalisée chez le rat. En conclusion, ce travail a permis de démontrer

l’application du concept d’ « emprisonnement » des composés biologiques dans des matrices

polymériques afin de les stabiliser et contrôler leur libération.
Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Drug carrier can improve drug utilization, safety and timeliness, reduce drug administration frequency, improve drug odor, improve dose accuracy and accurate drug release to targeted tissues and organs, so it has been widely concerned by people. The development of nanotechnology has promoted the research of drug carrier, and the research and application of nanoscale drug carrier has made great contributions in the field of medicine. In this paper, leucine (Leu) and hydroxyethyl starch (HES) were used to prepare nano-hydrogels, and Lau-Leu-HES nano-hydrogels were prepared to be used as drug carriers. The reaction method of 1- (3-dimethylaminopropyl) -3-ethylcarbondiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was used to promote condensation. We studied and characterized the appearance, molecular weight, particle size, pH response and other characteristics of the prepared nano-hydrogels. The performance of the two kinds of nano-hydrogels was studied by in vitro degradation experiment, drug-loading and drug-release experiment, and cytotoxic MTT experiment. The results showed that the prepared nano-hydrogels were suitable for drug carrier. In addition, we designed the factory for the production of the drug capsules and evaluated the entire process of quality control, risk assessment and solution for the conditions required to build the factory.
Носій активного фармацевтичного інгредієнту може поліпшити використання ліків, безпеку і своєчасність, зменшити частоту прийому препарату, поліпшити запах препарату, підвищити точність доз і точний викид препарату в цільові тканини і органи, тому це дуже актуальна тема. Розвиток нанотехнологій сприяє дослідженню носія лікарського засобу, а дослідження та застосування нанорозмірного носія лікарського засобу зробили великий внесок у галузі медицини. У цій роботі для приготування наногідрогелів використовувався лейцин (Лей) і гідроксиетиловий крохмаль (HES), а наногідрогелі Lau-Leu-HES були готові до використання в якості носіїв ліків. Метод реакції 1- (3-диметиламінопропіл) -3-етилкарбондіаміду гідрохлориду (ЕДК) і N-гідроксисучиніміду (NHS) був використаний для сприяння конденсації. Ми вивчили і охарактеризували зовнішній вигляд гелю, молекулярну масу, розмір частинок, реакцію на рН та інші характеристики підготовлених наногідрогелів. Продуктивність двох видів наногідрогелів була вивчена експериментом з деградації in vitro, експериментом з завантаженням та вивільнення лікарського засобу, а також дослідженням цитотоксичності MTT. Результати показали, що підготовлені наногідрогелі підходять як носії лікарських засобів. Крім того, ми розробили технологічний процес виробництва капсул препарату і оцінили весь процес контролю якості, оцінки ризиків і рішення для умов, необхідних для технологічного виробництва.

Titre : Nano-plaquettes à mobilité dirigée et adhésion amplifiée pour l’administration locale: vers un nouveau traitement des maladies vésicalesAbstract : L’administration locale des médicaments, définie comme une voie d’administration où la substance active est directement administrée sur ou proche de la cible ou tissus souhaités, permet d’apporter des grandes quantités des médicaments avec moins d’effets secondaires, et permet une simplification du système nanoparticulaire du fait de la non-extravasation des médicaments. Dans ce contexte, le projet de recherche de cette thèse s’est focalisé sur la voie intra-vésicale comme voie d’administration locale car il existe un besoin clinique de la part des patients, n’étant pas encore résolu. Malgré les hypothétiques avantages fournis par l’administration locale des médicaments, la voie intra-vésicale présente certaines limitations qui diminuent l’efficacité des traitements et l’observance des patients. La plupart des médicaments pour le traitement des maladies vésicales, notamment pour le cancer de la vessie et les cystites interstitielles, sont sous forme de solutions ou suspensions administrées de manière intra-vésicale via un cathéter qui passe à travers l’urètre. Dès leur arrivée à la vessie, les substances actives sont fortement diluées par les urines et éliminées rapidement lors de la miction. Cela conduit à une diminution des concentrations des substances actives au plus proche de l’épithélium, nécessitant plusieurs instillations intra-vésicales, réalisées par des praticiens hospitaliers, pour atteindre des concentrations thérapeutiques. Il y a donc un réel besoin de développer des nouvelles formulations permettant de contrecarrer les phénomènes décrits au préalable.L’objectif de cette thèse de doctorat est de créer un nouveau système nanoparticulaire de morphologie non-sphérique qui serait susceptible d’avoir un mouvement diffèrent et dirigé ainsi qu’une adhésion amplifiée. En conséquence, nous attendons de ces systèmes qu’ils apportent des concentrations en substances actives plus importantes que les systèmes nanoparticulaires sphériques et formulations galéniques traditionnelles.Aux cours de nos travaux expérimentaux, nous avons réussi à développer un système nanoparticulaire de morphologie hexagonale et aplatie. Ces nanoparticules, appellées nano-plaquettes, sont conçues à partir de l’auto-assemblage des molécules d’α-CD et des chaines alkyles greffées sur les squelettes de polysaccharides tels que l’acide hyaluronique, la chondroïtine sulfate ou l’héparine. Ces systèmes présentent l’originalité de ne pas avoir de substance active encapsulé parce que les molécules de polymère elles mêmes agissent à la fois en tant que substance active et de véhicule. Ces nano-plaquettes ont montré un mouvement en milieu isotrope et statique très diffèrent des nano-sphères utilisées comme contrôle. En effet, la majorité d’entre elles diffuse de manière plus importante et dirigée, avec des trajectoires rectilignes. Grâce à leur mouvement et aux propriétés inhérentes liées à leur forme, ces systèmes se sont montrés particulièrement intéressants vis-à-vis des interactions avec des cellules. Ils adhèrent mieux et plus longtemps à la muqueuse vésicale, elles sont mieux internalisées par des cellules et sont éliminées plus lentement une fois adhérées à la surface de l’urothélium.Un modelé in vivo de Syndrome de la Vessie Douloureuse / Cystite Interstitielle développé chez le rat nous a permis de montrer l’efficacité thérapeutique des nano-plaquettes, notamment celle constituées d’acide hyaluronique. En effet, elles présentent une meilleure bioaccumulation dans la vessie et une meilleure activité anti-inflammatoire et de régénération de la muqueuse urothéliale.Ces systèmes nanoparticulaires, conçues lors de nos travaux de thèse, constituent une approche innovante, rationnelle et efficace pouvant ouvrir de nouvelles voies de recherche pour le traitement des maladies vésicales
Title: Directed-mobility and enhanced-adhesion nano-platelets for local drug delivery: towards a new treatment of bladder diseases.Abstract: Local drug delivery, defined as the administration route where the drug is delivered directly or very close to its target or tissue, allows to bring large amounts of drugs with reduced side effects, in comparison with systemic administration. In this context, our research project has been focused on the intravesical drug delivery as local administration route, because there is a real need to develop new pharmaceutical formulations to thwart several limitations. Despite the advantages provided by the local drug delivery, intravesical drug delivery exhibited some issues which are decreasing the therapeutic efficacy and the patient compliance to the treatment. Most of therapies for the treatment of bladder diseases are simple drug solutions or suspensions administered intravesically by using a catheter through the urethra in order to reach easily the bladder and, consequently, the urothelium. Since the drug is administered into the bladder, drug dilution is occurring because the continuous production of urine. Furthermore, active substances are being eliminated during washout when bladder urine voiding is happening. These two processes lead to the decrease of local drug concentration close to the urothelium. Patients need repeated catheterization, performed by health care practitioners, to reach therapeutic dose of the drug. Therefor, there is a need of new drug formulations to avoid these main limitations.The main goal of this PhD thesis was to create and design a new nanoparticulate system with non-spherical shape susceptible to move in a different manner compared to spherical nanoparticles. These systems may exhibit an amplified mucoadhesion allowing to bring more important amounts of drug than classical and nanoparticle administration.During this thesis, we developed a new nanoparticulate system presenting non-spherical, hexagonal and flattened shape. The driven force for the design of these nanoparticles was the self-assembling of α-cyclodextrin molecules with alkyl chains grafted on the polymer skeleton. Polymers used belong to a polysaccharide family called glycosaminoglycans including hyaluronic acid, chondroitin sulfate or heparin. This original and innovative nanoparticulate system does not encapsulate an active drug. Our polysaccharide will act, at the same time, as the active drug and the carrier. These nanoparticles, called now nano-platelets have shown different movement behavior than the spherical ones. Indeed, they diffuse more rapidly in a straight-line way. Thanks to their oriented and directed motion and to their intrinsic properties, due to the shape, these systems have shown a better mucoadhesion on the bladder tissue, a better uptake in different cell lines and they were far less rapidly eliminated from the urothelium mucosa.An in vivo model of Bladder Painful Syndrome / Interstitial Cystitis in rats demonstrated the therapeutic efficacy of nano-platelets, especially for hyaluronic acid nanoparticles. Indeed, they demonstrated a better bioaccumulation into the bladder and a better therapeutic efficacy as anti-inflammatory and urothelium regenerating agents.These nanoparticulate systems, designed during this work, represent a new innovative, rational and effectiveness approach allowing to open new research pathways for the treatment of bladder diseases

A number of therapeutic proteins are used for long in clinical practice. These include for example insulin, calcitonine, growth hormone, and parathyroid hormone for the treatment of various systemic disorders, as well as protein antigens in vaccine formulations. Due to the recent developments in biochemical engineering and in the comprehension of the physiopathology of many diseases, peptides and proteins are expected to become a drug class of increasing importance. Recently, novel biological drugs have for example been developed such as monoclonal antibodies, antibody fragments, soluble receptors, and receptor agonists or antagonists. These are mainly used for the treatment of auto-immune and inflammatory diseases (asthma, rheumatoid arthritis) and for the treatment of cancers. However, a major drawback of these biomolecules is the need to use parenteral administration. This is mainly due to the harsh pH conditions that proteins undergo by oral administration, leading to various physico-chemical degradations and loss of biological activity.

Pulmonary delivery of these proteins could constitute an alternative to parenteral delivery. Due to the very high surface area of the lungs, the low thickness of the alveolar epithelium and the high level of lung vascularisation, pulmonary administration can indeed provide fast systemic absorption of drugs, while avoiding hepatic first pass metabolism. On the other hand, drugs for local treatment can also be administered directly into the lung, which allows delivering high doses while limiting systemic side effects. Nevertheless, administration of drugs to the lungs requires some challenges to be taken up. It is indeed necessary to provide the drug as very small solid or liquid microparticles (1-5 µm) in order to reach the lungs. For solid microparticles, it is also needed to overcome the very high inter-particle interactions by using appropriate formulation strategies and by including deaggregation mechanisms in the inhalation device. Other issues are more specifically related to the pulmonary administration of proteins. These can indeed undergo physico-chemical degradations during processing, administration, and/or storage. Moreover, if systemic action is required, proteins will often need addition of an absorption enhancer to cross the alveolar epithelium because of their large molecular weight and hydrophilicity.

In this work, we developed formulations for pulmonary delivery of proteins using two model proteins. Insulin (5.8 kDa) was chosen as a model of small protein. It is also an application of systemic pulmonary delivery. On the other hand, an anti-IL13 monoclonal antibody fragment (54 kDa) was used as a model of larger protein. This molecule is currently in development for the treatment of asthma and provided an application for local pulmonary delivery. The formulation strategy was to produce dry powders using a combination of micronisation techniques (high speed and high pressure homogenisations), drying techniques (spray-drying, freeze-drying), and addition of lipid excipients. These lipid excipients were added as a coating around the protein particles and were expected to prevent protein degradations during processing and/or storage, essentially by avoiding contact with water. It could also improve the aerodynamic properties of the powders by modification of the surface properties of the particles and/or limitation of the capillary forces.

First, we evaluated insulin lipid-coated formulations and formulations without excipients, produced using high pressure homogenisation and spray-drying. In the case of lipid-coated formulations, a physiological lipid composition based on a mixture of cholesterol and phospholipids was used. We were able to obtain good aerodynamic features for the different formulations tested, with fine particle fractions between 46% and 63% versus 11% for raw insulin powder. These are high FPF values in comparison with those obtained for other protein formulations for inhalation currently under development, which often have an in vitro deposition of around 30%. Insulin presented a good stability in the dry state, even when no lipid coating was added.

The presence of a lipid coating of up to 30% (w/w) did not significantly improve the aerodynamic behaviour of the powders, but the coated formulations exhibited decreased residual moisture content after 3-month storage, which should be of interest for the long-term stability of the formulations.

In a second step, two of the developed insulin formulations were evaluated in a clinical study to determine whether the formulations give high deep lung deposition in vivo, and how insulin is absorbed into the systemic blood stream. This pharmaco-scintigraphic trial was performed on twelve type 1 diabetic patients using an uncoated formulation and a formulation coated with 20% (w/w) of lipids. The two formulations showed interesting features, with pharmacokinetic profiles that mimic the natural insulin secretion pattern. Bioavailability was within the ranges of two of the three dry powder insulins that have reached phase III clinical development. However, the formulation with a lipid coating exhibited a lower lung deposition in comparison with the uncoated formulation, which was not expected from the previous in vitro results. Additional in vitro experiments indicated that this lower performance was related to a decrease in the disaggregation efficiency of the powder at a sub-optimal inhalation flow-rate. An extensive training of the patients to the inhalation procedure could therefore improve the lung deposition of the coated formulation.

Finally, we developed and evaluated dry powder formulations of the anti-IL13 antibody fragment. These were produced using, successively, freeze-drying, high pressure homogenisation (HPH), and spray-drying. The influence of different types and concentrations of stabilising excipients was evaluated for each production step. Due to its more elaborated structure, the antibody fragment was found to be more sensitive than insulin to physico-chemical degradation, particularly during the HPH process, which led to different types of degradation products. These could partly be avoided by adding 50% sucrose during freeze-drying and 10% Na glycocholate or palmitic acid in the liquid phase during HPH (dispersing agents). However, the presence of a small fraction of insoluble aggregates could not be fully avoided. Further spray-drying of the suspensions in the presence of 10% Na glycocholate or palmitic acid led to the formation of a hydrophilic or hydrophobic coating around the particles, respectively. Na glycocholate was found to be particularly effective in protecting the antibody during spray-drying, which was found to be at least partly related to its ability to inhibit sucrose recrystallisation. However, the best formulation still presented a small fraction of insoluble aggregates (6%). The aerodynamic evaluation of the formulations showed FPFs that were compatible with lung deposition, with the formulation containing Na glycocholate presenting the highest FPF (42%). The formulation coated with palmitic acid presented a slightly lower FPF (35%). The aerodynamic properties of this formulation remained unchanged at a sub-optimal inspiratory flow rate, to the contrary of what was observed for the insulin formulation coated with 20% (w/w) cholesterol and phospholipids. Palmitic acid could therefore be of interest as a hydrophobic coating material, and provide long-term stability of protein drugs.

The work performed with the insulin and anti-IL13 molecules provided the proof-of-concept that it was possible to obtain dry powder protein formulations with appropriate aerodynamic properties and good overall physico-chemical stability, using simple production techniques and few selected excipients. The formulation strategy presented in this work could therefore be of interest for the future development of inhaled proteins for local or systemic applications.

Doctorat en sciences pharmaceutiques
info:eu-repo/semantics/nonPublished