Dissertations / Theses on the topic 'Drug nanoparticles'

Wet bead milling, in which the drug is milled in presence of stabilisers such as polymers and surfactants, has enabled the formulation of poorly water-soluble drugs as nanoparticles, with five products having reached the market. During the milling process, the polymer and/or surfactant adsorbs onto the freshly cleaved drug surfaces to provide ionic or steric stabilisation. Despite the success of wet bead milling, mastery of the mechanism behind nanoparticle stabilization is still lacking. To investigate whether any relationship exists between drug, stabiliser and stabilisation, eight structurally different poorly water-soluble drugs were milled in presence of thirteen different pharmaceutically acceptable stabilisers and the resultant particle size determined by photon correlation spectroscopy. Nanoparticles of the BCS class II drugy griseofulvin, could only be produced in presence of anionic stabilisers namely sodium dodecyl sulphate, aerosol-OT or hydroxypropylmethylcellulose acetate succinate. Surfactant adsorption isotherms obtained indirectly by measuring their depletion from solution revealed a maximum surfactant adsorption of ~ 2.2 mg/m2 on the griseofulvin nanoparticle surfaces. The use of ionic surfactants/polymers in oral formulations is however sub-optimal. Consequently, polymer-surfactant co-stabilisation, used to take advantage of the synergy between ionic and non-ionic stabilisers, was investigated by the inclusion of the non-ionic polymer hydroxypropylmethylcellulose (HPMC) into the anionic surfactant-drug slurry prior to milling. The effect of varying HPMC molecular weight and concentration on griseofulvin nanoparticle production was established. Polymer adsorption isotherms were obtained directly via small angle neutron scattering.

Although various drugs have been developed to treat different diseases such as cancer, the therapeutic effects of many drugs have been limited by their undesirable properties such as poor solubility, poor bioactivity, rapid clearance in blood and non-specific distribution. Nanoparticles as carriers have received more and more attention in the last two decades due to their ability of overcoming these obstacles and enhancing the therapeutic efficiency of the conventional drugs. In this thesis, various kinds of nanoparticles were developed aiming at improving the therapeutic efficiency and targeted delivery of anti-cancer drug and gene. Curcumin is a promising anti-cancer drug but its applications in cancer therapy are limited due to its poor solubility, short half-life and low bioavailability. In this thesis, magnetic-polymer core-shell nanoparticles based on non-toxic, biocompatible and biodegradable polymers such as silk fibroin, alginate and chitosan were prepared and optimized to improve the uptake efficiency and cell growth inhibition effect of curcumin towards cancer cells. The size, zeta potential, surface morphology, drug loading / release profile, in vitro uptake and growth inhibition effect to cancer and normal cells of these curcumin loaded nanoparticles were investigated. The results indicated that the curcumin loaded particles exhibited enhanced uptake efficiency and growth inhibition effect on MDA-MB-231 cancer cells compared with free curcumin. Higher uptake efficiency and cytotoxicity to MDA-MB-231 cells than normal human dermal fibroblast cells were observed, suggesting they have specific effects against cancer cells. Moreover, in vitro targeted delivery of curcumin to specific areas of cells was achieved with the presence of an external magnetic field, suggesting these magnetic nanoparticles are promising for targeted delivery of drugs to desired sites applying magnetic forces. Apart from drug delivery the applications of magnetic nanoparticles in gene delivery was also investigated. Polyethyleneimine is one of the most efficient non-viral transfection agents for gene delivery due to its high cationic charge density. In this thesis, silk fibroin was selected to fabricate magnetic-silk / polyethyleneimine core-shell nanoparticles and silk-polyethyleneimine nanoparticles for the transfection of an anticancer gene (c-myc antisense oligodeoxynucleotides) into MDA-MB-231 breast cancer cells and human dermal fibroblast cells. The results illustrated that the cytotoxicity of magnetic-silk / polyethyleneimine core-shell nanoparticles was significantly lower than polyethyleneimine coated magnetic nanoparticles which is widely studied as a gene delivery carrier. The magnetic-silk / polyethyleneimine core-shell nanoparticles were capable of delivering c-myc antisense oligodeoxynucleotides into MDA-MB-231 cells and significantly inhibiting the cell growth. Employing magnetic-silk / polyethyleneimine core-shell nanoparticles, high uptake efficiency of c-myc antisense oligodeoxynucleotides was achieved within 20 min via magnetofection. In addition, magnetic-silk / polyethyleneimine core-shell nanoparticles exhibited higher cytotoxic effect against MDA-MB-231 breast cancer cells than normal human dermal fibroblast. Moreover, in vitro targeted delivery of oligodeoxynucleotides can be achieved using magnetic-silk / polyethyleneimine core-shell nanoparticles under a magnetic field.

L’ús de nanopartícules (NPs) ha emergit com una eina prometedora pel tractament de càncer. Entre els seus usos en teràpia i en tècniques d’imatge, destaca l’ús com agents de direccionament de fàrmacs. Tot i així, encara es requereix un profund coneixement d’aquests sistemes abans de poder aplicar-los a la clínica. Aquí presentem l’ús de nanopartícules d’or (AuNPs) per a detoxificar un dels fàrmacs de major ús en quimioteràpia, el cisplatí. Aquest fàrmac està lligat a la NP via un enllaç de coordinació sensible a pH per assegurar l’alliberament de fàrmac als endosomes. El tamany de les NPs juga un paper molt important en determinar algunes respostes biològiques com la biodistribució o la seva remoció per part del sistema immune. Per això, és indispensable controlar perfectament el tamany de la NP en la seva síntesis abans de qualsevol aplicació biològica. Aquí descrivim un nou protocol per sintetitzar AuNPs amb un control exquisit del tamany entre 5 i 200 nm. Un dels avantatges d’aquest protocol és l’obtenció de NPs estabilitzades amb citrat que poden ser funcionalitzades a posteriori. D’aquesta manera podem aprofundir aquí en el mecanisme de formació de monocapes autoensamblades (SAM) i capes formades per barreges de surfactants. El control pel que fa a la composició i conformació d’aquestes és molt important doncs determina respostes biològiques com l’adsorció de proteïnes i l’estabilitat col·loïdal en medis fisiològics. Aquests conjugats han servit com a esquelet per enllaçar-hi cisplatí via la formació d’un enllaç de coordinació que asseguri un alliberament de la droga desencadenat per una baixada de pH. Aquesta conjugació està caracteritzada en profunditat per tal de garantir la estabilitat col·loïdal així com la de l’enllaç. Finalment, el disseny del conjugat té efectes significatius en les propietats farmacocinètiques, en l’evolució del propi conjugat i en la manca de toxicitat. En aquest treball mostrem en models animals com la toxicitat deguda a cisplatí disminueix clarament sense afectar això a les propietats terapèutiques del fàrmac. A més a més, les NPs no només actuen com a vehicles sinó que també protegeixen el fàrmac contra la inactivació per part de proteïnes del sèrum fins que els conjugats són internalitzats per cèl·lules i el cisplatí alliberat. La possibilitat de seguir el fàrmac (Pt) i el vehicle (Au) separadament en funció de l’òrgan i el temps aporta també un millor coneixement sobre com els nanovehicles són processats per l’organisme.
The use of nanoparticles (NPs) has emerged as a potential tool to improve cancer treatment. Among the proposed uses in imaging and therapy, their use as a drug delivery scaffold has been extensively highlighted. However, there are still some controversial points which need a deeper understanding before applying them in the clinics. Here, it is presented the use of gold nanoparticles (AuNPs) to detoxify the antitumoral agent cisplatin linked to the nanoparticle via a pH sensitive coordination bond for endosomal release. Since size of NPs plays an important role in determining biological responses such as biodistribution or clearance by immune system, a perfect control on the synthesis of AuNPs is required previously to any biological application of these AuNPs. It is described in this work a new synthetic protocol of biocompatible AuNPs with a perfect control of the size between 5 to 200 nm. One of the advantages of this protocol is the obtaining of citrate-capped AuNPs that can be further functionalized. This allowed us to provide insights on the mechanism of Self-Assembled Monolayers and mixed layers formation. The control of the mixed layer composition and conformation is important since it determines biological outcomes such as protein adsorption and colloidal stability in physiological media. These AuNPs conjugates are used as scaffold for cisplatin attachment via the formation of a coordination bond that ensures a pH-triggered release of the drug. This conjugation is deeply characterized to ensure the maintenance of colloidal and link stability on working conditions. Finally, the NP conjugate design has important effects on pharmacokinetics, conjugate evolution and biodistribution and absence of observed toxicity. Here we show that cisplatin-induced toxicity is clearly reduced without affecting the therapeutic benefits in mice models. The NPs not only act as carriers, but also protect the drug from deactivation by plasma proteins until conjugates are internalised in cells and cisplatin released. Also, the possibility to track the drug (Pt) and the vehicle (Au) separately as a function of organ and time enables a better understanding of how nanocarriers are processed by the organism.

Des microorganismes produisant des molécules tensioactives ont été isolés à partir d’échantillons de sols contaminés par des huiles, en provenance des provinces de Songkhla et Chiangmai (Thaïlande) et de Shianghai (Chine). Les différentes souches ont été sélectionnées de façon à obtenir les biosurfactants ayant les meilleures propriétés tensioactives et d’émulsification. Parmi 102 souches isolées, 6 microorganismes produisaient des biosurfactants. La souche SK80 a conduit aux meilleures propriétés tensioactives. Des observations morphologiques macroscopiques et microscopiques ont permis de caractériser la souche SK80. L’analyse de la séquence ARNr 28S indique que cette souche appartient à la famille Exophiala Dermatitidis. La composition du milieu de culture (source de carbone et d’azote) et les conditions de culture de ce microorganisme ont été adaptées de façon à obtenir des quantités importantes de biosurfactant. Des analyses spectroscopiques (RMN 1H, RMN 13C, COSY et de masse, APCI MS) ont révélé que ce biosurfactant était un monooléate de glycérol. La monomyristine a été choisie comme constituent synthétique modèle dans des études d’encapsulation. Deux méthodes de préparation, émulsion/évaporation de solvant, nanoprécipitation, ont été employées pour encapsuler la monomyristine dans des nanoparticules recouvertes de dextrane et dont le cœur était constitué de poly(acide lactique) ou de dextrane hydrophobisé. Les conditions d’encapsulation ont été variées afin de maximiser le rendement d’encapsulation et la stabilité colloïdale des particules
Biosurfactant producing microorganisms were isolated from oil contaminated soils collected from Songkhla and Chiangmai province, Thailand and Shianghai, China. Their culture broths were screened for obtaining biosurfactants with the highest surface activity and emulsification ability. Among 102 isolates, 6 microorganisms produced biosurfactants. The culture supernatant of SK80 strain exhibited the highest surface activity. SK80 was identified by macroscopic morphology, microscopic morphology and showed that it is a black mold. The 28S rRNA sequence homology analysis suggested that SK80 belongs to Exophiala dermatitidis. The composition of culture medium such as carbon source, nitrogen source, and culture condition of this microorganism was optimized to obtain high amounts of biosurfactant. 1H NMR, 13C NMR, COSY and Mass Spectrometer (APCI MS) results indicated that this biosurfactant was monoolein (oleoyl glycerol), a kind of monoacylglycerol. Monomyristin was chosen as a monoacylglycerol model to be synthesized and used as nanoparticle encapsulated drug. Two preparation methods, emulsion/solvent evaporation and nanoprecipitation, were used to encapsulate monomyristin in dextran-covered nanoparticles with poly(lactic acid) of hydrophobized dextran as the core material. Encapsulation conditions were optimized with regard to the yield encapsulation and the colloidal stability

Apoptosis (programmed cell-death) is a common final pathway through which cells die in retinal degenerative diseases. The purpose of this project was to develop biodegradable nanoparticles that quickly deliver XIAP, an inhibitor of apoptosis, to retinal cells following acute insults. In vitro protein release profiles from different formulations were established, and two cell types were incubated with nanoparticles to assess cellular uptake. Subretinal injections were carried out in rats to assess in vivo localization and possible toxicity. In vitro studies showed an initial burst of protein followed by sustained release, with overall low levels of protein release. Cell culture experiments suggest that particles are mostly membrane-bound, and some may be internalized. In vivo experiments revealed no signs of toxicity, and protein localized within the photoreceptor layer. In conclusion, nanoparticles may provide a good delivery system for XIAP; however higher levels of protein release are needed for neuroprotection, warranting further investigation.

Thesis (Ph. D.)--Ohio State University, 2004.
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We demonstrate intracellular delivery of various molecules by inducing controlled and reversible cell damage through pulsed laser irradiation of carbon black (CB) nanoparticles. We then characterized and optimized the system for maximal uptake and minimal loss of viability. At our optimal condition 88% of cells exhibited uptake with almost no loss of viability. In other more intense cases it was shown that cell death could be prevented through addition of poloxamer. The underlying mechanism of action is also studied and our hypothesis is that the laser heats the CB leading to thermal expansion, vapor formation and/or chemical reaction leading to generation of acoustic waves and then there is energy transduction to the cell causing poration of the cell membrane. We also delivered anti-EGFR siRNA to ovarian cancer cells. Cells exposed to a laser at 18.75 mJ/cm2 for 7 minutes resulted in a 49% knockdown of EGFR compared to negative control. We established an alternative way to deliver siRNA to knockdown proteins, for the first time using laser CB interaction.

A major challenge of cancer treatment is successful discrimination of cancer cells from healthy cells. Nanotechnology offers multiple venues for efficient cancer targeting. Magnetoelectric nanoparticles (MENs) are a novel, multifaceted, physics-based cancer treatment platform that enables high specificity cancer targeting and externally controlled loaded drug release. The unique magnetoelectric coupling of MENs allows them to convert externally applied magnetic fields into intrinsic electric signals, which allows MENs to both be drawn magnetically towards the cancer site and to electrically interface with cancer cells. Once internalized, the MEN payload release can be externally triggered with a magnetic field. MENs uniquely allow for discrete manipulation of the drug delivery and drug release mechanisms to allow an unprecedented level of control in cancer targeting. In this study, we demonstrate the physics behind the MEN drug delivery platform, test the MEN drug delivery platform for the first time in a humanized mouse model of cancer, and characterize the biodistribution and clearance of MENs. We found that MENs were able to fully cure the model cancer, which in this case was human ovarian carcinoma treated with paclitaxel. When compared to conventional magnetic nanoparticles and FDA approved organic PLGA nanoparticles, MENs are the highest performing treatment, even in the absence of peripheral active targeting molecules. We also mapped the movement through peripheral organs and established clearance trends of the MENs. The MENs cancer treatment platform has immense potential for future medicine, as it is generalizable, personalizable, and readily traceable in the context of treating essentially any type of cancer.

Existeix un interès creixent, tant en el món acadèmic com en la recerca industrial en el desenvolupament de sistemes d’alliberament de fàrmacs macromoleculars (proteïnes, pèptids, oligonucleotids) capaços de travessar la mucosa. En aquest sentit, la utilització de vectors sintètics per a l’alliberament de les esmentades macromolècules, permet disposar d’una plataforma versàtil i altament eficient. Tanmateix, la capa de mucosa amb propietats adhesives i altament viscoelàstica, té una elevada capacitat d’atrapar i eliminar qualsevol substància estranya que quedi adherida sobre la seva superfície, limitant, de forma evident, la eficàcia de qualsevol tractament Aquesta Tesi es centra en el desenvolupament de sistemes d’alliberament de DNA, dissenyats a mida, que presenten una elevada estabilitat i eficàcia de transfecció amb un nivell molt baix de toxicitat i molt important en el context de la tesi, una capacitat de permeació a través de la mucosa. A més la tesi també es centra en el disseny i el desenvolupament de mètodes i tècniques in vitro que ajudin a una millor selecció de sistemes eficients d’alliberament a través de la mucosa. Així s’ha desenvolupat un mètode simple i eficient, basat en la utilització de una microbalança de quartz amb dissipació (QCM-D). Aquest mètode ha permès avaluar la interacció de polímers i nanopartícules amb una capa de mucina. Els resultats obtinguts amb el mètode desenvolupat han permés dissenyar sistemes de nanopartícules amb un potencial més gran de permeació a través de la mucosa. Aquesta tècnica d’alta sensibilitat també ha ofert la possibilitat d’avaluar las dos propietats oposades, el coneixement de les quals és necessari per un correcte disseny de sistemes cpaços de creuar la mucosa: mucoadhesió vs mucopenetració. Los Poly(β-amino ester)s (PBAEs) s’han proposat com a sistemes biodegradables capaços de formar nanopartícules, per complexació amb DNA, que presenten una elevada capacitat de transfecció. Tanmateix, mostren problemes d’estabilitat en condicions fisiològiques i són incapaços de travessar la capa de mucosa. En aquesta tesi es descriu una nova solució en la preparació de les formulacions dels nanocomplejos basada en la utilització de recobriments que estabilitzen les nanopartícules i augmenten la seva permeabilitat. Els recubrimeintos proposats inclutyen: i) sucres (sucroses, trhalosa i manitol), ii) quitosà sense modificar de 22 KDa i amb 60-120 kDa, iii) quitosan modificat amb àcid tioglicolidoi i iv) acid poliacrílic-bromelaina. Totes les noves formulacions s’han avaluat amb diferents quantitat de recobriment. S’han determinat les seves propietats fisicoquímiques i la seva eficàcia de transfecció i citotoxicitat en front de cèl.lules COS-7. S’ha estudiat La difusió de las partícules a través de la mucosa gàstrica de porc utilitzant diferents tècniques com el tub rotatori de silicona o el multiple particle tracking (MPT). Els resultats obtinguts han mostrat la superior estabilitat, eficàcia de transfecció i permeabilitat sobre la mucosa de las noves formulacions dissenyades.
Existe un interés creciente, tanto en el mundo académico como en la investigación industrial en el desarrollo de sistemas de liberación de fármacos macromoleculares (proteínas, péptidos, oligonucleótidos) capaces de atravesar la mucosa. En este sentido, la utilización de vectores sintéticos para la liberación de dichas macromoléculas, permite disponer de una plataforma versátil y altamente eficiente. Sin embargo, la capa de mucosa con propiedades adhesivas y altamente viscoelástica, tiene una elevada capacidad de atrapar y eliminar cualquier sustancia extraña que quede adherida sobre su superficie, limitando, de forma evidente, la eficacia de cualquier tratamiento Esta Tesis se centra en el desarrollo de sistemas de liberación de ADN, diseñados a medida, que presentan una elevada estabilidad y eficacia de transfección con un nivel muy bajo de toxicidad y muy importante en el contexto de la tesis, una capacidad de permeación a través de la mucosa. Además la tesis también se centra en el diseño y el desarrollo de métodos y técnicas in vitro que ayuden a una mejor selección de sistemas eficientes de liberación a través de la mucosa. Así se ha desarrollado un método simple y eficiente, basado en la utilización de una microbalanza de cuarzo con disipación (QCM-D). Este método ha permitido evaluar la interacción de polímeros y nanopartículas con una capa de mucina. Los resultados obtenidos con el método desarrollado han permitido diseñar sistemas de nanopartículas con un mayor potencial de permeación a través de la mucosa. Esta técnica de alta sensibilidad también ha ofrecido la posibilidad de evaluar las dos propiedades opuestas, el conocimiento de las cuales es necesario para un correcto diseño de sistemas cpaços de cruzar la mucosa: mucoadhesió vs mucopenetració. Los Poly (β-amino ester)s (PBAEs) se han propuesto como sistemas biodegradables capaces de formar nanopartículas, por complejación con ADN, que presentan una elevada capacidad de transfección. Sin embargo, muestran problemas de estabilidad en condiciones fisiológicas y son incapaces de atravesar la capa de mucosa. En esta tesis se describe una nueva solución en la preparación de las formulaciones de los nanocomplejos basada en la utilización de recubrimientos que estabilizan las nanopartículas y aumentan su permeabilidad. Los recubrimeintos propuestos inclutyen: i) azúcares (sucrosa, trhalosa y manitol), ii) quitosano sin modificar de 22 KDa y con 60-120 kDa, iii) quitosano modificado con ácido tioglicólico y iv) ácido poliacrílico-bromelaina. Todas las nuevas formulaciones se han evaluado con diferentes cantidades de recubrimiento. Se han determinado sus propiedades fisicoquímicas y su eficacia de transfección y citotoxicidad frente a células COS-7. Se ha estudiado La difusión de las partículas a través de la mucosa gástrica de cerdo utilizando diferentes técnicas como el tubo rotatorio de silicona o el múltiple particle tracking (MPT). Los resultados obtenidos han mostrado superior estabilidad, eficacia de transfección y permeabilidad sobre la mucosa de las nuevas formulaciones diseñadas.
Mucus penetrating nanoparticle-based delivery systems of macromolecular drugs are currently receiving increasing attention in both academic and industrial research. Synthetic delivery systems provide highly suitable and tunable platform for the delivery of the macromolecules. However, a highly viscoelastic and adhesive mucus layer generally traps and rapidly removes most foreign substance from the mucosal surfaces, thereby limiting effectiveness of these nanocarriers. This Thesis is addressed to the development of engineering DNA delivery systems capable of high stability and transfection efficiency with low toxicity, and quickly crossing the mucus layer. Moreover, this Thesis is focused on design and development of methods and techniques in vitro in order to select more efficient delivery systems. A simple and efficient method, based on the use of the quartz crystal microbalance with dissipation (QCM-D) technique, is developed and evaluated the interaction of the polymers and nanoparticles with the mucin layer, resulting in the development of nanoparticle-based delivery systems to mucosal tissue. This highly sensitive technique also offers to evaluate the two opposing properties, needed for the design of efficient mucous permeation systems: mucoadhesion vs mucus penetration. Poly(β-amino ester)s (PBAEs) are currently considered of great interest as biodegradable polymeric carriers of DNA delivery, but they present limited stability in physiological conditions and the inability to penetrate the mucus layer. In this Thesis, we describe a novel surface-modified formulation of DNA delivery systems consisting of PBAE/DNA complexes and the coating agents, including: i) sugars (sucrose, trehalose or mannitol), ii) unmodified chitosan with a 22 kDa (CS) and a with a 60-120 kDa (CSM), iii) chitosan-thioglycolic acid (CS-TGA), and iv) poly(acrylic acid)-bromelain (PAA-BRO) conjugates. All novel formulations formed with different amounts of the coating agents are evaluated the physicochemical properties. The influence of coating agents on transfection efficiency and cytotoxicity is evaluated in COS-7 cells. Particle diffusion through porcine intestinal mucus (PImucus) is assessed by either rotating silicone tube technique or multiple particle tracking (MPT). The results highlight the superior stability, transfection efficiency and mucus permeability of the novel nanoparticle-based drug delivery systems. The effect of the amount of coating agents is also discussed.

En la present tesi doctoral, s'ha desenvolupat un sistema d'alliberament de fàrmacs sensible a pH basat en nanopartícules mesoporoses de sílice (MSN). Els porus de la nanopartícula estan radialment obstruïts mitjançant la funcionalització de cadenes de PEG, substituïdes amb un fàrmac en un dels seus extrems. L'objectiu d'aquesta nova metodologia és la de preservar la càrrega interna d'aquestes MSN. En primer lloc, s'ha estudiat el concepte d'obstrucció radial per avaluar la utilitat pràctica d'aquest mètode. Per aquesta raó, diferents tipus de cadenes de PEG amb càrrega, a saber, amines quaternàries i PEG neutrals, s'han funcionalitzat sobre la MSN per estudiar la seva capacitat d'obstrucció. Com a prova de concepte, s'ha estudiat l'alliberament de safranina en medi fisiològic (pH 7.4). Els resultats obtinguts han demostrat que les cadenes de PEG que contenen una càrrega positiva obstrueixen millor els porus que les cadenes de PEG neutres de la mateixa longitud. Utilitzant aquesta aproximació, s'ha dissenyat un sistema d'alliberament de fàrmacs per a la vehiculització de camptotecina (CPT) i topotecan (TPT). En primer lloc, un dels anteriors fàrmacs s’ha adsorbit dins dels porus de la MSN. Posteriorment, els porus s’han segellat mitjançant una cadena de PEG que conté doxorrubicina (DOX) en un dels seus extrems (DOX-PEG). L'estabilitat d'aquest sistema en condicions fisiològiques prova l'eficàcia de l'obstrucció radial. D'altra banda, en condicions àcides, es produeix un alliberament descontrolat dels fàrmacs. Així mateix, els experiments de citotoxicitat in vitro han demostrat que el sistema pot alliberar CPT i DOX en les cèl·lules cancerígenes HeLa, aconseguint un major efecte sinèrgic que la combinació de TPT i DOX. També s'ha sintetitzat un profàrmac de la CPT amb l'objectiu d'augmentar la seva càrrega en una MSN i millorar així el seu efecte sinèrgic amb la DOX. Per dur-ho a terme, s’ha unit una cadena de PEG escindible a la CPT. Emprant aquesta estratègia s'ha aconseguit carregar un 30% més de CPT a l'interior de les MSN. El sistema mostra una gran estabilitat en condicions fisiològiques, ja que s’observa un alliberament negligible dels fàrmacs. A més, s'ha avaluat la citotoxicitat de sistema en dues línies cel·lulars diferents: HeLa i HepG2. Els resultats obtinguts demostren que el nou profàrmac sintetitzat amb combinació amb la DOX té un major efecte sinèrgic en les cèl·lules HepG2. D'altra banda, la selectivitat de les MSN cap a les cèl·lules HepG2 s'ha millorat mitjançant la funcionalització sobre el grup DOX-PEG amb el lligant àcid glicirretinic (GA). Per fer-ho, s'ha emprat la mateixa aproximació radial establerta pels altres sistemes. Els estudis d'internalització cel·lular han demostrat que aquest nou sistema és capaç de discriminar entre cèl·lules HeLa i HepG2, acumulant-se preferentment en aquestes últimes. Finalment, s'ha avaluat un sistema d'administració triple de medicaments amb l'objectiu de superar l'efecte de resistència dels tumors a múltiples fàrmacs. Aquesta acció es pot emprendre mitjançant la combinació d’agents quimioterapèutics, DOX i CPT, amb un agent fototerapèutic (ftalocianina). Referent a això, s'ha sintetitzat un nou conjugat de la CPT amb una ftalocianina. Aquest conjugat s'ha carregat dins dels porus de la MSN i posteriorment s'han segellat amb el grup DOX-PEG. Els experiments d'internalització cel·lular han demostrat l'endocitosi d'aquest sistema en les cèl·lules HeLa i el posterior alliberament dels fàrmacs. Així mateix, s'ha avaluat in vitro l'efecte sinèrgic entre la DOX i la CPT.
En la presente tesis doctoral, se ha desarrollado un sistema de liberación de fármacos sensible a pH basado en nanopartículas mesoporosas de sílice (MSN). Los poros de la nanopartícula están radialmente obstruidos mediante la funcionalización de cadenas de PEG, sustituidas con un fármaco en uno de sus extremos. El objetivo de esta nueva metodología es la de preservar la carga interna de estas MSN. En primer lugar, se ha estudiado el concepto de obstrucción radial para evaluar la utilidad práctica de este método. Por esta razón, diferentes tipos de cadenas de PEG con carga, a saber, aminas cuaternarias y PEG neutrales, se han funcionalizado sobre la MSN para estudiar su capacidad de obstrucción. Como prueba de concepto, se ha estudiado la liberación de safranina en medio fisiológico (pH 7.4). Los resultados obtenidos han demostrado que las cadenas de PEG que contienen una carga positiva obstruyen mejor los poros que las cadenas de PEG neutrales de la misma longitud. Utilizando esta aproximación, se ha diseñado un sistema de liberación de fármacos para la vehiculización de camptotecina (CPT) y topotecán (TPT). En primer lugar, uno de los anteriores fármacos se ha adsorbido dentro de los poros de la MSN. Posteriormente, los poros se han sellado mediante una cadena de PEG que contiene doxorrubicina (DOX) en uno de sus extremos (DOX-PEG). La estabilidad de dicho sistema en condiciones fisiológicas prueba la eficacia de la obstrucción radial. Por otro lado, en condiciones ácidas, se produce una liberación descontrolada de los fármacos. Asimismo, los experimentos de citotoxicidad in vitro han demostrado que el sistema puede liberar CPT y DOX en las células cancerígenas HeLa, logrando un mayor efecto sinérgico que la combinación de TPT y DOX. También se ha sintetizado un profármaco de la CPT con el objetivo de aumentar su carga en una MSN y mejorar así su efecto sinérgico con la DOX. Para llevarlo a cabo, se ha unido una cadena escindible de PEG a la CPT. Empleando esta estrategia se ha conseguido cargar un 30% más de CPT en el interior de las MSN. El sistema muestra una gran estabilidad en condiciones fisiológicas, ya que se observa una liberación negligible de los fármacos. Además, se ha evaluado la citotoxicidad del sistema en dos líneas celulares diferentes: HeLa y HepG2. Los resultados obtenidos demuestran que el nuevo profármaco sintetizado en combinación con DOX, resulta en un mayor efecto sinérgico en las células HepG2. Por otro lado, la selectividad de las MSN hacia las células HepG2 se ha mejorado mediante la introducción del ligando ácido glicirretínico (GA) sobre el grupo DOX-PEG. Para llevarlo a cabo, se ha empleado la misma aproximación radial establecida para los otros sistemas. Los estudios de internalización celular han demostrado que este nuevo sistema es capaz de discriminar entre las células HeLa y HepG2, acumulándose preferentemente en estas últimas. Finalmente, se ha evaluado un sistema de administración triple de medicamentos con el objetivo de superar el efecto de resistencia de los tumores a múltiples fármacos. Esta acción se puede emprender mediante la combinación de medicamentos quimioterapéuticos, DOX y CPT, con un agente fototerapéutico (ftalocianina). Por esta razón, se ha sintetizado un nuevo conjugado de la CPT con una ftalocianina. Dicho conjugado se ha cargado dentro de los poros de la MSN y posteriormente se han sellado con el grupo DOX-PEG. Los experimentos de internalización celular han demostrado la endocitosis de este sistema en las células HeLa y la posterior liberación de los fármacos. Asimismo, se ha evaluado in vitro el efecto sinérgico entre la DOX y la CPT.
In this PhD dissertation, a pH-responsive multiple drug delivery system (DDS) based on mesoporous silica nanoparticles (MSN) with a radial-capping of its pores has been developed. This is a new concept that relies on the functionalization of the particle surface with PEG chains substituted with a drug at its end to preserve the inner cargo of the MSN. First, the concept of radial-capping has been studied to assess the practical usefulness of such capping method. Thus, different types of charged PEGs, namely quaternary amines and neutral PEGs, have been introduced upon an MSN in order to study its capping ability. As a proof of concept, the dye safranin was loaded into the nanoparticles pores, which were subsequently capped with PEGs chains. Then, the release of safranin was assessed under physiological conditions (pH 7.4). The results obtained demonstrated that PEG chains possessing positive charge provides a more efficient capping than the neutral PEGs of the same length. Using this approach, a drug delivery system (DDS) based on the radial capping for the delivery of camptothecin (CPT) and topotecan (TPT) has been studied. CPT or TPT has been loaded within the pores of an MSN, and subsequently sealed with a PEG chain decorated with doxorubicin (DOX) at its end (DOX-PEG moiety). The system is stable under physiological conditions (pH 7.4) which confirms the effectiveness of the radial capping. On the other hand, under acidic pH, a burst release of drugs takes place. Furthermore, the in vitro cytotoxicity test has demonstrated that this DDS can effectively deliver CPT and DOX to HeLa cells achieving a better synergistic effect than the combination of TPT and DOX. With the aim to improve the loading of CPT to enhance the synergistic effect with the latter system (DOX-PEG moiety), a prodrug of CPT has been synthesised. To do so, a cleavable reductive short PEG chain has been bonded to CPT. An increase of loading of 30% has been achieved in comparison with the unmodified drug. The stability of the radial-capping methodology has been tested as mentioned above. Under physiological conditions, the release of drugs is negligible. The cytotoxicity activity of the system has been tested in two different cell lines: HeLa and HepG2 cells. The results showed a better synergistic effect of this new synthesised system towards HepG2 cells. In order to further improve the selectivity of the system towards HepG2 cells, the MSN were decorated with glycyrrhetinic acid (GA) ligand over the DOX-PEG moiety. Uptake studies have shown that this new system preferably accumulates in HepG2 cells in comparison to HeLa cells. Finally, a tri-deliver system of drugs has been developed with the aim to try to overcome the multiple drug-resistant (MDR) effect by the combination of chemotherapeutic drugs (DOX and CPT) with a phototherapeutic agent (phthalocyanine). In this regard, a new CPT conjugate with a phthalocyanine has been synthesised and loaded within the pores of an MSN. Then, the system has been sealed with the DOX-PEG moiety. The uptake studies have demonstrated the proper endocytosis of the system inside HeLa cells and the subsequent delivery of the three drugs in the cytoplasm and nucleus. Furthermore, the synergistic effect of DOX and CPT has been assessed in vitro.

Hydrogel micro- and nanoparticles (microgels and nanogels) are a promising class of drug delivery vehicles. Composed of hydrophilic polymers arranged into a cross-linked network structure, nanogels show several attractive features for the delivery of macromolecule therapeutics. For instance, the hydrated, porous internal cavity of the nanogel may serve as a high capacity compartment for loading macromolecules, whereas the periphery of the nanogel may be used as a scaffold for conjugating cell-specific targeting moieties. This dissertation presents recent investigations of nanogels as targeted delivery vehicles for oligonucleotides to cancer cells, while exploring new nanogel chemistries that enable future in vivo applications. For instance, synthetic efforts have produced particles capable of erosion into low molar mass constituents, providing a possible mechanism of particle clearance after repeated administration in vivo. In another example, the microgel network chemistry was tuned to promote the encapsulation of charged proteins. In parallel with those synthetic efforts, new light scattering methodologies were developed to accurately quantify the particle behaviors (e.g. loading, erosion). Using multiangle light scattering (MALS), changes in particle molar mass and radius were measured, providing a quantitative and direct approach for monitoring nanogel erosion and macromolecule encapsulation. The new particle chemistries demonstrated, together with enabling light scattering methods, will catalyze the development of improved delivery vehicles in the near future.

The aim of this work was the design and characterisation of drug-loaded, polymeric targeting nanoparticles (NP). Poly(lactide-co-glycoIide) (PLGA), a well known biodegradable and biocompatible polymer, was used for NP preparation. NP were formulated using controllable diffusion and emulsification combinations. This procedure was able to produce NP with narrow particle size distribution, high drug entrapment efficiency and ability to sustain drug release. To achieve drug targeting, ligand was conjugated to the NP surface using carbodiimide chemistry. The density of reactive carboxyl groups on the surface of PLGA NP was modulated by combining high molecular weight, end-capped PLGA, RG 505 S with a low molecular weight, non end-capped PLGA, RG 502 H. Such apprC?ach was able to conjugate protein-type targeting ligand onto NP surface along with modulation of drug release profile. Increasing the RG 502 H proportion in PLGA blend was found to increase the amount of ligand conjugation to NP surface as well as rate of drug release. Anti-Siglec-7 polyclonal antibody directed toward an endocytotic receptor was conjugated to the NP surface. Confocal laser scanning microscopy images obtained with Nile Red-loaded NP and fluorescence. microscopy images obtained with Acridine Orange-loaded NP, suggested selective binding of anti-Siglec- 7 conjugated NP to cells expressing Siglec-7 receptor with possible intracellular uptake. The ability of cytotoxic drug-loaded targeting NP to improve cytotoxicity was evaluated by encapsulating camptothecin, topoisomerase-I inhibitor, into PLGA NP with different densities of surface carboxylic acid, which was then conjugated with IgG anti-Fas monoclonal antibody. In vitro antitumour activity, evaluated using human colorectal cancer cell lines (HCT116), indicated that PLGA NP were able to improve CPT anti-tumour activity compared with CPT solution. Conjugating anti-Fas mAb onto the surface of CPT-loaded NP resulted in improved potency compared to corresponding naked NP, and was able to synergise with CPT.

Towards the development of drug carriers that are capable of crossing the Blood Brain Barrier, the techniques of emulsion polymerisation and nanoprecipitation have been utilised to produce nanoparticulate carriers from a systematic series of alkylglyceryl dextrans (of two different average molecular weights, 6 kDa and 100 kDa) that had been functionalised with ethyl and butyl cyanoacrylates. Also, zero length grafting of polylactic acid to butyl, octyl and hexadecylglyceryl dextrans has allowed the preparation of polylactic acid-functionalised nanoparticles. All materials and derived nanoparticles have been characterised by a combination of spectroscopic and analytical techniques. The average size of nanoparticles has been found to be in the range 100-500 nm. Tagging or loading of the nanoparticles with fluorophores or model drugs allowed the preliminary investigation of their capability to act as controlled-release devices. The effects of an esterase on the degradation of one such nanoparticulate carrier have been studied. Testing against bend3 cells revealed that all materials display dose-dependent cytotoxicity profiles, and allowed the selection of nanocarriers that may be potentially useful for further testing as therapeutic delivery vehicles for conditions of the brain.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2008.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Vita.
Includes bibliographical references (p. 193-208).
Therapeutics such as proteins, DNA, or siRNA, can only exert their function in the cell cytosol or nucleus. However, most of them are cell membrane impermeable molecules that can only be taken up by cells via endocytosis or phagocytosis. Such drug molecules are thus confined in endolysosomes, where reduced pH and degradative enzymes may destroy them without therapeutic gain. Efficient escape of drug molecules to the cytosol before destruction in endolysosomes is a major challenge for intracellular drug delivery. To address this issue, we designed a pH-sensitive core-shell nanoparticle to segregate the functions of the particle into an endosome-disrupting pH-responsive core that would absorb protons at endolysosomal pH, and a shell whose composition could be tuned to facilitate particle targeting, cell binding, and drug binding. Two-stage surfactant-free emulsion polymerization of 2-diethylamino ethyl methacrylate (DEAEMA) (core) and 2-amino ethyl methacrylate (AEMA) (shell) in the presence of a crosslinker was used for the synthesis of monodisperse core-shell hydrogel nanoparticles of 200 nm in diameter. The protonation of tertiary amine groups on the polyDEAEMA core on moving from extracellular to endolysosomal pH resulted in reversible swelling of the nanoparticles with a 2.8-fold diameter change. With the aid of pH-sensitivity of these nanoparticles, efficient cytosolic delivery of calcein (with ~95% efficiency) was achieved by disrupting endolysosomes via proton sponge effect. The primary amine rich shell was found to facilitate cell and drug binding, and provided negligible cytotoxicity by sequestering the proton sponge component from any direct interactions with cells. These particles demonstrated a useful means to deliver therapeutic molecules to the cytosol of cells of interest efficiently.
(cont.) The applications of nanoparticles showed significant improvement in delivering a model antigen vaccine protein ovalbumin (OVA) to primary dendritic cells for T cell activation, and promising knockdown of mRNA by delivering siRNA to epithelial cells for gene silencing. To extend this approach to a fully biodegradable system, nanoparticles with a cleavable crosslinker bis (acryloyl) cystamine (BAC) were synthesized. Preliminary explorations of this approach showed that such particles can degrade in the presence of glutathione in vitro, a reducing peptide present at mM concentrations in the cytosol of mammalian cells. This design could potentially serve as a drug releasing mechanism to further improve delivery efficiency.
by Yuhua Hu.
Ph.D.

In recent years inorganic nanoparticles have been of great scientific interest not only due to the fact that they can be tailored in morphology allowing for the tuning of their optical and electronic properties but also due to their rich surface chemistry. Recent advances in conjugation techniques have allowed for the surface functionalization of nanoparticles with ligands such as synthetic oligonucleotides. This has led to the development of bio-nanomaterials that have been successfully used in applications ranging from bio sensing to the targeted delivery of molecules such as drugs. This thesis focuses on the design and synthesis of advanced DNA-coated gold nanostructures that can perform the synergistic tasks of sensing and drug delivery in cells. Gold nanoparticles functionalized with synthetic oligonucleotides were assembled into dimers using copper free click chemistry and were used for the intracellular detection of up to two mRNA targets. Once taken up by cells they showed great biocompatibility, no significant susceptibility to degradation by nucleases and most importantly excellent specificity towards the mRNA sequence they were designed to detect. Furthermore, DNA-coated gold nanoparticle dimers were developed into multifunctional nanostructures. In addition to live cell mRNA detection we showed how they could also be designed to deliver up to two chemotherapeutic drugs. By relying on the specificity of the gold nanoparticle dimer towards an mRNA target, cell specific drug delivery was made possible thus demonstrating the synergistic capabilities of this system. Finally with a view of shedding light on the interaction between DNA-coated gold nanoparticles and cells their intracellular fate including the time point and location of mRNA detection after cellular entry was investigated.

Thesis (M.S.)--University of New Orleans, 2004.
Title from electronic submission form. "A thesis ... in partial fulfillment of the requirements for the degree of Master of Science in the Department of Chemistry."--Thesis t.p. Vita. Includes bibliographical references.

Delivery of complex molecules such as peptides, proteins, oligonucleotides and plasmids is an intensively studied subject, which has attracted considerable medical and pharmaceutical interest. Encapsulation of these molecules with biodegradable polymers represents one way of overcoming various problems associated with the conventional delivery of macromolecules, for example instability and short biological half-life. The use of carriers made of hydrophilic polysaccharides such as chitosan, has been pursued as a promising alternative for improving the transport of biologically active macromolecules across biological surfaces. The development of nanoparticles as a delivery system also has major advantages of achieving possible drug protection, controlled release and drug targeting by either a passive or an active means. The aim of this study was to develop a simple and effective method to formulate biodegradable nanoparticles for the delivery of a model protein-bovine serum albumin (BSA) and an angiogenesis inhibitor, arginine-rich hexapeptide (ARE peptide). Major factors which determine nanoparticle formation and loading of the protein and the peptide as well as the underlying mechanisms controlling their incorporation and release characteristics were investigated. The preparation technique, based on the complex coacervation process, is extremely mild and involves the mixture of two aqueous solutions (chitosan and dextran sulfate) at room temperature. The formation of nanoparticles is dependent on the concentrations of chitosan (CS) and dextran sulfate (DS); particles with size, of 257 to 494nm can be obtained with 0.1%w/v solutions of CS and DS. Zeta potential of nanoparicles can be modulated conveniently from -34.3mV to +52.7mV by varying the composition of the two ionic polymers.Both bovine BSA and the ARH peptide were successfully incorporated into CS-based nanoparticles, mainly via an electrostatic interaction, with entrapment efficiency up to 100% and 75.9% for the protein and peptide respectively. Incorporation of both the protein and peptide into nanoparticles resulted in an increase in size suggesting their close association with the nanoparticle matrix material. The difference in sign and magnitude of zeta potential of empty and macromolecules-loaded nanoparticles supports the hypothesis that protein and peptide association with nanoparticles can be modulated by their ionic interaction with the oppositely charged ionic polymer (DS) in the nanoparticles. The release of BSA from the nanoparticles was very slow in water compared to that in l0mM phosphate buffer pH 7.4; whereas, ARH peptide showed extremely low level of release in water at the low ratio of DS but at the high ratio of DS, its release was in biphasic fashion, with an initial burst effect followed by an almost constant but very slow release up to 7 days in both water and 1 OmM phosphate buffer (pH 7.4). It was found that, unlike ARH peptide, the percentage of BSA released was relatively slower for the nanoparticles with a high ratio of DS. It is speculated that this difference in the release behaviour of BSA and ARH peptide, could be due to the effect of molecular size of the compounds and their interaction with the polymer matrix of the nanoparticle. The results of this study suggest that these novel CS/DS nanoparticulate system, prepared by a very mild ionic crosslinking technique, have potential to be a suitable carrier for the entrapment and controlled release of peptides and proteins.

PLGA have already been successfully applied for controlled drug delivery systems by the pharmaceutical industry due to its biocompatibility, biodegradability and ease of processing. It has recently further been developed and formulated into a form of nanoparticle. The single emulsion evaporation method was used to prepare nanoparticles in this study. By varying different parameters such as the concentration of regents, the type of surfactant and emulsion method, different particle sizes and size distribution of PLGA nanoparticles could be obtained. The stability of PLGA nanoparticles was further investigated by assessing their thermal property over a certain period of time using DSC. The decrease of Tg confirmed the hydration and degradation of PLGA polymers and nanoparticles. The changes of surface morphology showed that the nanoparticles were in spherical shape and maintained smooth surface before the storage, whereas they started to lose their original shapes as well as agglomerate to each other after 2-week storage. These results suggested that there was an erosion and degradation of PLGA nanoparticles during storage. Ibuprofen-loaded PLGA nanoparticles have been successfully prepared by o/w single emulsion evaporation method. During the stability study, a faster degradation rate compared to non-loaded PLGA nanoparticles was exhibited, showing that Ibuprofen increased the degradation rate of PLGA nanoparticles. According to the results of drug releasing study, PLGA nanoparticles exhibiting a slower drug release rate than pure drug which proved that drug-nanoparticule system could effectively increase the stability of drugs. PLGA polymer is a potential material for drug delivery system.

Amorphous solid dispersions of polysaccharide-drug nanoparticles were produced by a rapid precipitation process known as flash nanoprecipitation and the formulation process and properties of nanoparticles were investigated. In this thesis, several novel cellulose derivatives and a pullulan derivative were studied. Among these polymers, carboxymethyl cellulose acetate butyrate (CMCAB)-drug nanoparticles were investigated in detail. Previous work has shown that the presence of different chemical groups in CMCAB could aid in complexation with hydrophobic drugs with low solubility, forming an amorphous matrix which can increase the effective solubility and, hence, bioavailability of the drug in physiological conditions. An antibacterial drug and two less soluble anti-viral drugs were selected as model drugs for this study. A separate study was conducted with several other cellulose derivatives like cellulose acetate propionate adipates with two different degree of substitution 0.33 and 0.85 (CAP-Adp 0.33 and CAP-Adp 0.85), cellulose acetate sebacate (CA-320S Se) and butyl pullulan-6-carboxylate (BPC) polymers. The effect of polymer interaction with drug molecule on release of antiviral drugs was studied with these latter polymers.
    The purpose of this research was two-fold. First, the methodology for producing drug-polymer nanoparticles with well-defined particle size distributions was developed. Second, the factors affecting drug loading and release properties of these nanoparticles were investigated. The nanoparticles were processed using two methods of solvent removal and drying to investigate their effects on drug loading and particle size: (a) various combinations of rotary vacuum evaporation (rotavap) and acid-induced flocculation were used and (b), dialysis followed by freeze drying. Dynamic light scattering showed particle sizes were between 150-400 nm with polydispersity index values as low as 0.12. The antibiotic drug loading efficiencies ranged from 14-40%, whereas drug loading efficiency as high as 85 % was attained with the antiviral drug. The dissolution studies showed an increase in the solution concentration and release of the amorphous drug nanoparticles. The high glass transition temperature helped to stabilize the drug in an amorphous form, thus increasing the effective solution concentration of the drug in an aqueous medium.

Ph. D.

Une méthodologie générale et efficace pour la synthèse de nanoparticules de prodrogues polymères hétérotéléchéliques à hauts taux de charge a été mise au point en combinant d’une part la méthode dite du “principe actif amorceur” pour obtenir des prodrogues polymères α-fonctionnelles par polymérisation radicalaire contrôlée par les nitroxydes (NMP), et d’autre part la réaction d'échange de nitroxyde à partir d’un nitroxyde fonctionnel pour coupler une seconde molécule d'intérêt en bout de chaîne. Une petite bibliothèque de prodrogues polymères hétérotéléchéliques avec différentes combinaisons pour diverses applications (e.g., libération de principes actifs, imagerie/théranostic, thérapie combinée, ciblage actif) a été synthétisée en utilisant le polyisoprène (PI) comme polymère.En particulier, une alcoxyamine basée sur le nitroxyde SG1 a été fonctionnalisée avec la première molécule d’intérêt et utilisée pour polymériser l'isoprène par NMP et donner la prodrogue polymère désirée. En appliquant ensuite la réaction d'échange de nitroxyde à partir du nitroxyde TEMPO fonctionnalisé avec la seconde molécule d’intérêt, le nitroxyde SG1 en bout de chaîne a été quantitativement remplacé par le TEMPO fonctionnel pour donner la prodrogues hétérobifonctionnelle. Cette approche générale a été appliquée aux combinaisons suivantes : (i) gemcitabine (Gem)/rhodamine (Rho) et Gem/cyanine pour la libération de principes actifs et l’imagerie; (ii) aminoglutethimide (Agm)/doxorubicine (Dox), Gem/Dox and Gem/Lapatinib (Lap) pour la thérapie combinée et (iii) Gem/biotine pour la libération de principes actifs et le ciblage actif in vitro et in vivo. Les propriétés d’imagerie des nanoparticules de prodrogues polymères comportant une molecule fluorescente ont été étudiées in vitro et in vivo, respectivement en termes d’internalisation intracellulaire et de biodistribution. Pour les thérapies combinées, la cytotoxicité in vitro des différentes nanoparticules a été étudiée et comparée à celle émanant d’autres strategies de délivrance de deux principes actifs (e.g., conanoprécipitation, mélange physique de nanoparticules).Enfin, des prodrogues polymères hétérobifonctionnelles comprenant l’adénosine en début de chaîne et un motif maléimide en fin de chaine ont été préparées et fonctionnalisées en surface par des protéines capables de promouvoir le passage des nanoparticules à travers la barrière hémato-encéphalique pour libérer l’adénosine au niveau du cerveau
A facile and versatile synthetic platform to prepare high drug loading, heterobifunctional polymer prodrug nanoparticles was developed by combining the “drug-initiated” method to obtain α-functional polymer prodrugs by nitroxide-mediated polymerization (NMP), and the nitroxide exchange reaction from a functional nitroxide to attach a second molecule of interest at the ω chain-end. A library of heterotelechelic polymers prodrugs with different combinations for various purposes (e.g., drug delivery, imaging/theranostic, combination therapy, active targeting) was prepared using polyisoprene (PI) as polymer scaffold. More specifically, an alkoxyamine based on the SG1 nitroxide was functionalized with the first drug of interest and used to perform the NMP of isoprene to yield the desired polymer prodrug. Subsequently, by applying the nitroxide exchange reaction using a TEMPO nitroxide functionalized with the second molecule of interest, the SG1 nitroxide at the chain-end was quantitatively replaced by the functional TEMPO and the desired heterobifunctional polymer prodrug was formed. This general methodology was applied to the following combinations: (i) gemcitabine (Gem)/rhodamine (Rho) and Gem/cyanine for drug delivery and imaging; (ii) aminoglutethimide (Agm)/doxorubicin (Dox), Gem/Dox and Gem/Lapatinib (Lap) for combination therapy and (iii) Gem/biotin for drug delivery and active targeting in vitro and in vivo. For polymer prodrug nanoparticles bearing fluorescent dyes, in vitro and in vivo imaging studies were performed to investigate their cellular internalization and their biodistribution, respectively. As for the different combination therapies, the in vitro cytotoxicity of the nanoparticles was determined and compared to that of other strategies to deliver two different drugs (e.g., conanoprecipitation, physical mixture of nanoparticles).Finally, heterobifunctional polymer prodrugs bearing adenosine in alfa position and a maleimide moiety in omega position were synthesized to give nanoparticles that were further surface-functionalized with different proteins able to promote crossing through the blood brain barrier for drug delivery to the brain

The limited access to the brain of a large number of therapeutic actives due to the presence of the blood-brain barrier (BBB) has led to intensive research toward the development of nanotechnology-based approaches. Polysaccharides such as chitosan, guar gum, pectin and pullulan have been selected as starting materials for this study due to their biocompatibility, biodegradability, good drug carrier properties, and ease of chemical modification with short chain alkylglycerol-like moieties (expected to enhance drug permeability through the BBB). A series of butylglyceryl-modified polysaccharides were prepared and characterised using chromatographic, spectroscopic and thermal analysis techniques prior to formulation into nanoparticles (NPs) by means of a selection of methods that include reverse emulsification, nanoprecipitation, and ionotropic gelation. Dynamic Light Scattering, Nanoparticle Tracking Analysis, Electrophoretic Mobility Measurements and Electron Microscopy were employed to characterise all NPs (overall size range 120–200 nm, and zeta potential values ranging from -27 to +39 mV). Modified pullulan (PUL-OX4) and guar gum (GG-OX4) NPs were found to be most stable at physiological pH (7.4), in contrast to chitosan (CS-OX4) NPs that demonstrated an increase in size as a result of aggregation. PUL-OX4 NPs (< 145 nm) had the highest Angiotensin II model peptide loading (8.46 %), while GG-OX4 NPs showed the highest loading degree with Doxorubicin (19.11 %) and Rhodamine B (3.78 %). Drug release studies demonstrated that PUL-OX4 NPs released fastest all the model actives tested, while GG-OX4 NPs were able to retain them for the longest period of time. The in vitro interactions of NPs with mouse brain endothelial cells (bEnd3) were investigated using a Transwell permeability model, with results suggesting an increased model membrane permeability in the presence of the modified polysaccharide nanoparticles. The cytotoxicity of these NPs at physiologically-relevant concentrations was studied using MTT assays; all NPs were non-toxic at concentration below 2 mg/mL, however a decrease in cell viability was noticed at higher concentrations. PUL-OX4 nanoparticles were found to be the least toxic, having the lowest LC50 value (9.48 mg/mL; for comparison, CS-OX4 has 7.30 mg/mL). Haemolysis study demonstrated that at concentration below 12 mg/mL, all the NPs studied did not induce a haemolysis effect significantly when compared to PBS control, however an increase in the effect was observed at higher concentration. PUL-OX4 nanoparticles exhibited the highest LC30 value of 19.87 mg/mL while the lowest value was exhibited by CS-OX4 nanoparticles (13.95 mg/mL). Confocal microscopy and flow cytometry investigations confirmed that all modified polysaccharide NPs were successfully taken up by bEnd3 cells, becoming localised in the cytoplasm.

Within the last decades, the stated potential of polymer constructs as drug delivery systems have challenged researchers to develop sophisticated polymers with tunable properties. The versatility of polymers makes them highly attractive to tailor nanoparticles (NPs) which fulfill the demands of effective drug delivery systems (DDS). The aim of this work was to design and synthesize amphiphilic ethylene glycol methacrylate-based (EGMA) macromolecules, and explore their potential as NPs for drug delivery. Initially, a study of the controlled synthesis and solution properties of linear EGMA polymers, as well as the potential to transfer their behavior to amphiphilic comb copolymers, was conducted. Well-controlled polymers with interesting tunable thermo-responsive properties were accomplished by altering the monomer feed ratio. Furthermore, the comb copolymers formed self-assembled core-shell type structures in aqueous solution. A library of amphiphilic fluorinated polymers was successfully established to explore the potential of EGMA-based polymers in a dual-functional theranostic delivery system. The non-toxic polymers self-assembled into small “stealthy” NPs, and the combination of fluorinated segments with EGMA segments allowed for detection by 19F-MRI with good imaging properties. The hydrophobic core of the NPs was capable to encapsulate and release an anti-cancer therapeutic, and effectively reduced the viability of three different cancer cell lines. The diffusion-controlled release kinetics of the drug from the NPs interestingly depended on the nature of the core moiety. To reduce issues with instability of self-assembling NP systems the possibility to synthesize amphiphilic hyperbranched dendritic-linear polymers (HBDLPs) was investigated. Their three-dimensional structure was hypothesized to facilitate stabilization as unimolecular micelles. The architecture, hydrophilic/hydrophobic ratio, and high molecular weight showed to be crucial to avoid polymer association and stabilize the HBDLPs individually. In addition, the hyperbranched core of the HBDLPs was readily functionalized with disulfide bonds, either in the backbone or in the pendant groups. Under reductive conditions, selective cleavage of the disulfides thereby enabled either significant molecular weight reduction, or allowed for triggered release of a covalently bound dye, mimicking a drug. Potentially, such HBDLPs could be stable during circulation, while allowing for selective degradation and/or therapeutic release upon delivery to a cancer tissue.

QC 20150909

The main aim of this study is to synthesize theranostic nanoparticles (NPs) that will drastically increase the diagnostics and therapeutic efficacy for cancer. In this research, we had prepared the NPs which constitute carbon dots (CDs), the imaging agent, Folic acid, the targeting agent, and Doxorubicin (DOX) or Gemcitabine (GEM) as the chemotherapy agents. The prepared NPs include noncovalent FA-CDs-DOX, covalent CDs-FA-DOX, and covalent FA-CDs-GEM. The spectroscopy, ultraviolet-visible spectroscopy (UV-vis), fluorescence spectroscopy, and Fourier transform-infrared spectroscopy (FT-IR), were used to confirm the successful fabrication of these complexes. Through UV-vis analysis, the drug loading capacity (DLC) and drug loading efficiency (DLE) of the complexes were determined. The noncovalent series had a higher DLE of about 83% while the covalent series showed higher DLC, 70% on average indicating high drug content. The in-vitro pH-dependent drug release shows that the noncovalent FA-CDs-DOX and the covalent FA-CDs-GEM series release more drugs into the cancer cells (pH of 5.0) than into healthy normal (pH of 7.4). The sizes of NPs were measure around 2-5 nm with Dynamic light Scattering (DLS). The toxicity of CDs, CDs-drug, and FA-CDs-drug on MDA-MB468 breast cancer cell was tested through the methylthiazolytetrazolium (MTT) assay and found that the FA bonded NPs exhibited strong therapeutic efficacy. More pharmaceutical data towards the cancer cells are investigated by our research collaborators – the pharmaceutical department at ETSU and Xavier University at Louisiana.

This study aims to prepare theranostic nanoparticles (NPs) that are expected to increase cancer diagnostics and therapeutic efficacy. We prepared the NPs constituting carbon dots (CDs) as an imaging agent, folic acid as a targeting agent, doxorubicin (DOX), or gemcitabine (GEM) as chemotherapy agents. The NPs include noncovalent FA-CDs-DOX, covalent CDs-FA-DOX, and covalent FA-CDs-GEM. Through ultraviolet-visible spectroscopy, fluorescence spectroscopy, and Fourier transform-infrared spectroscopy, the fabrication of these NPs was confirmed. It was discovered that the high drug loading efficiency is the noncovalent series while the high drug loading capacity is the covalent series The in-vitro pH-dependent drug release data indicate the NPs release more drugs at around pH 5.0 than at pH 7.4. The NPs sizes are between 2-5 nm. The Cell viability was investigated using the Alamar Blue assay and the three NPs complexes exhibited strong therapeutic efficacy against MDA-MB-468 breast cancer cells as compared with CDs-drug.

In this project an overview of how synthetic lipid nanoparticles and exosomes can be used for targeted drug delivery is compiled. The goal is to identify aspects that can be in favor for targeted drug delivery and the development of products at Cytiva. The most important fields for Cytiva to understand is the methods and the challenges of cell culturing for production of exosomes, productions of lipid nanoparticles, purification of exosomes, analysis of both exosomes and lipid nanoparticles, and how exosomes and lipid nanoparticles are used as tools for drug delivery. To understand these aspects a description focusing on structural components, specific delivery and cargo loading is also included in the report. Many different components and methods have been found in the different fields mentioned, and the ones that we believe are the most relevant for Cytiva are presented and discussed in the report. We conclude that both exosomes and lipid nanoparticle are suitable options as drug delivery vehicles, especially for their ability to be modified for targeted delivery, encapsulate therapeutic compounds and cross biological barriers. Exosomes are also biostable and possess low immunogenicity. For production the methods identified with highest potential are Hollow-Fiber Bioreactor for cell culturing in production of exosomes and Microemulsion and High-Pressure Homogenization for lipid nanoparticles. Purification is required for exosomes and the most prominent method is Size-Exclusion Chromatography, because of its scalability. After production and purification it is important to be able to detect the vesicles and the most developed and used methods are Nanoparticle Tracking Analysis and Flow Cytometry, beacuse they can use labeling techniques and single vesicle analysis.

The methanolic atom transfer radical polymerisation (ATRP) of 2-hydroxypropyl methacrylate (HPMA) to controllably form the hydrophobic polymer p(HPMA) using a one-pot methodology at ambient temperature has been demonstrated, where polymerisations were shown to reach >99 % conversion. By simple variation of initiator:monomer feed ratio, polymers of varying chain length were synthesised. Using identical polymerisation conditions, addition of a small amount of ethylene glycol dimethacrylate (EGDMA) divinyl brancher resulted in the generation of high molecular weight branched copolymers without any modification of reaction kinetics. This approach was extended to include the first synthesis of linear and branched amphiphilic A-B block copolymers using polyethylene oxide (PEG) macroinitiators without loss of the ATRP controlled polymerisation. A series of systematically varying copolymers, containing variation in PEG length and/or variation in p(HPMA) primary chain length, have been synthesised to allow direct comparison of the impact of architectural variation on polymer properties. Nanopreciptation approaches were investigated for the linear and branched copolymers and extremely stable hydrophobic nanoparticles were produced using copolymers with branched architecture. Moreover, it has been shown that nanoparticle z-average diameter can be controlled using extremely facile methods. The loading capacity of amphiphilic branched A-B block copolymer nanoparticles with various guest-molecules has been systematically investigated. The world leading HIV/AIDS antiretroviral drug Lopinavir (LPV) was used in a preliminary loading screen and shown to produce candidate LPV/drug nanocarrier options for future studies and optimisation.

Nanoparticles were synthesized and modified for target drug delivery. The research involved the aqueous synthesis of near infrared (NIR) sensitive Au-Au₂S nanoparticles. An anti-cancer drug (cis-platin) was subsequently adsorbed onto the Au-Au₂S nanoparticle surface via the 11-mercaptoundecanoic acid layers. The results showed that the degree of adsorption of cis-platin onto Au-Au₂S nanoparticles was controlled by the pH value of solution, and the rate of drug release was sensitive to NIR irradiation. The results of the synthesis, drug-release properties and nanoparticle-cell interactions will be discussed.
Singapore-MIT Alliance (SMA)

Nanoparticles are important tools in biotechnology and biomedical research. Gold nanoparticles (AuNPs) have emerged as a particularly important class of nanobiotechnological tools as a result of a number of unique and useful attributes. These attributes include the high degree of biocompatibility of AuNP cores, the similarity in size of AuNPs and biomacromolecules, and the great chemical flexibility of AuNP surface design. One of the most promising applications of AuNPs in biotechnology and biomedicine is their use as drug delivery vehicles. Drug delivery vehicles provide therapeutics with desired delivery properties by targeting them specifically to the environments in which their therapeutic activity is sought and by overcoming solubility barriers. The drug delivery properties of AuNPs are a function of their sizes and surface chemistries. The nanometer scale of AuNPs allows these three-dimensional and diffusible self-assembled monolayers to act as substructures for supramolecular assemblies, to extravasate from tumor-supplying endothelia, and to undergo cellular uptake by endocytosis. AuNPs have become a versatile platform for the creation of multifunctional delivery vehicles. This work represents a collection of studies in which AuNPs have been used as probes in fundamental biological research and delivery systems for small molecules and biologics. In these studies, precision control of surface chemistry on the nanometer scale, made possible by AuNPs, has been used to find solutions to the problems of unraveling the role of hydrophobicity in immune system activation, delivering proteins past mammalian cell membranes, development of a sustained release drug delivery platform, and condensation and cellular delivery of siRNA.

The goal of the present work is to synthesize chitosan-coated superparamagnetic iron oxide nanoparticles (CS SPIONs) for doxorubicin (DOX) delivery for cancer theranostics. The CS SPIONs will be loaded with the anticancer drug DOX because it is largely used clinically for different cancers types. In this work chitosan nanoparticles (CS NPs) and iron oxide nanoparticles were synthetized by ionic gelation and thermal decomposition techniques, respectively. Chitosan depolymerization was performed to be used different molecular weights (474 – 39 kDa) to produce CS NPs with different diameters. Magnetic stirring and pH influence were also studied. Dynamic Light Scattering (DLS) measurements indicate that was obtained different nanoparticles diameters, approximately the lowest diameters were around 100 nm and 9 nm for CS NPS and iron NPs respectively. Then, CS SPIONs were formed. Synthetized nanoparticles were characterized by (DLS), UV-Visible (UV-Vis), Fourier Transform Infrared Spectroscopy (FTIR) and Transmission Electrons Microscopy (TEM). Superconducting Quantum Interference Device (SQUID) and magnetic hyperthermia studies indicate that this nanoparticles show a superparamagnetic behavior and the ability to generate heat. These characteristics are essential to be possible to use these nanoparticles in biomedical applications such as contrast agents for MRI, magnetic drug delivery, cancer diagnostics and treatment. The DOX delivery studies indicate that the drug release depends on pH and in the first 10-20 hours the majority of drug is released. Finally, the in vitro cell viability and proliferation studies were conducted using the Vero cell line. These studies indicate that the CS SPIONs synthetized in the present work are non-toxic up to the CS SPIONs concentration of 1.25 mg/ml. Considering all the studies conducted in this work, it can be concluded that the nanoparticles synthetized possess the necessary characteristics to be used in biomedical applications.