Tesis sobre el tema "Drug-Polymer conjugates"

Polymer drug conjugates are an emerging class of therapeutic agent that offer advantages in the treatment of cancer through long circulation times and passive targeting. A further benefit of using a polymeric framework is that it readily allows attachment of targeting motifs to enhance the drug specificity, as well as allowing variation of the drug component. In this work a series of targeted polymer drug conjugates were prepared that incorporated different drugs via peptide linkers designed to be stable in circulation but degradable at the target site. In order to ensure stability in circulation, it was necessary to ensure the drugs were conjugated to the biodegradable linkers via amides, which required the introduction of amine functionalities to the natural-product drugs. The required hemisyntheses were successfully carried out, although yields were sometimes disappointing. An effort to allow an assessment of the pharmacokinetic effects of using a polymeric scaffold was made through the attempted synthesis of low molecular weight analogues of the polymer drug conjugates, with success in some cases. A recently developed polymeric precursor was used in the preparation of the polymer drug conjugates but was found to undergo an unexpected side-reaction, which may prevent the long term development of this particular precursor. The desired conjugates were nonetheless successfully produced on a scale suitable for initial biological testing. Both the high and low molecular weight constructs synthesised showed reduced cytotoxicities to P388 murine leukemia cells and are currently awaiting in vitro testing to truly evaluate their utility.

PEGylation has become very popular for the generation of nanomedicines with improved protein delivery properties, despite its lack of biodegradability. Researchers usually try to maximise retained protein activity during PEGylation. However, this proof of principle study aimed to create an inactive peptide or enzyme product, using a biodegradable polymer, that would elicit minimal activity/non-specific toxicity on administration. Following triggered site-specific degradation of the polymer, the hypothesis was that protein activity could be slowly regenerated in the general circulation or localised to a specific target site. Model conjugates were synthesised by coupling dextrin degraded by amylase to trypsin and melanocyte stimulating hormone MSH, to test this concept and targeted delivery for both an enzyme and a receptor-binding ligand. Hyaluronic acid HA degraded by hyaluronidase conjugates of trypsin and ribonuclease A were also synthesised. The latter was intended to develop the possibility of designing novel anti cancer conjugates. A higher molecular weight dextrin 47,200 g/mol, 26 mol succinoylation was shown to best mask 34 trypsin activity and reinstate 58 of the activity by addition of amylase. When a HA fraction molecular weight 130,000 g/mol was prepared by acid hydrolysis and conjugated to trypsin 4 w/w, trypsin activity was masked to 6 and immediately re-instated to 24 on addition of hyaluronidase. Similarly, the dextrin-MSH conjugate reduced melanin production to 11 of the control and only restored to 33 on addition of amylase. RNase A alone was not cytotoxic up to 1 mg/mL, whereas, the HA-RNase A conjugate 0.1 mg/mL RNase A equivalent was cytotoxic in B16F10 and CV-1 cells 72 h. This work provides proof of principle for the concept of using biodegradable polymers to mask and reinstate conjugated protein activity in the presence of the appropriate enzyme 'unmasking' trigger.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2005.
Includes bibliographical references.
We have conceptualized a new class of polymer-linker-drug conjugates to achieve targeted drug delivery for the systemic treatment of cancer and other inflammatory diseases. The physiochemical properties of the polymer allow the conjugate to circulate longer in the body by minimizing renal and hepatic clearance, thereby improving the pharmacokinetics of the attached drugs. Traditionally, linkers are degraded by acidity or by some ubiquitous intracellular enzymes. We incorporate linkers that are sensitive to a specific extracellular enzyme whose overexpression is co-localized with the diseased tissue. The drug molecules remain inactive when attached to the polymer, thus preventing normal tissues from harmful side effects. When the conjugate is transported to the diseased area where there is a high level of the target enzyme, the linkers are cleaved to release the drugs at the specific site. As an example, we designed and synthesized two generations of novel polymer-peptide-drug conjugates for the tumor-targeted delivery of chemotherapeutics. To allow for passive targeting and enhanced permeation and retention (EPR), dextran with a size greater than 6 nm was selected as the polymeric carrier. This biocompatible and biodegradable carrier was chemically modified to allow for conjugation with doxorubicin and methotrexate, two common chemotherapeutics with undesirable side effects.
(cont.) Since matrix-metalloproteinases (MMPs) are associated with a number of types of cancer and their functions are essential to disease progression, including degrading extracellular matrix, releasing angiogenic factors and activating growth factors, we explored the possibility of MMP-mediated drug release. The synthesis procedures combined solid phase and solution phase techniques to enable flexibility in the linker design and in the charge modification of the polymer. This scaleable and robust process produced new conjugates that demonstrated excellent stability under physiological conditions and optimized sensitivity to enzymatic cleavage by MMP-2 and MMP-9. The new conjugate, dextran-peptide-methotrexate, was assessed for its in vivo anti-tumor efficacy and systemic side effects. It was compared to free methotrexate and a similar conjugate, differing by an MMP-insensitive linker, at equivalent intraperitoneal dosages administered weekly. The MMP-sensitive conjugate resulted in effective inhibition of in vivo tumor growth in each of the two separate tumor models that overexpress MMP-2 and MMP-9 (HT-1080 and U- 87). In contrast, free methotrexate resulted in no significant tumor reduction in the same models. Neither free methotrexate nor the conjugate caused any tumor inhibition in mice bearing RT- 112, a slower-growing model which expresses significantly less MMP than HT-1080 and U-87 . The anti-proliferative effect of the drug contributed to the inhibition of tumor growth. Systemic side effects caused by the MMP-sensitive conjugates were tolerable.
(cont.) MMP-insensitive conjugates, though able to inhibit tumor growth, caused toxicity in the small intestine and bone marrow and the experiment was terminated after one injection. We conducted a biodistribution study in HT-1080 bearing mice to investigate the targeting mechanism of the new conjugate. Independent of the linker sequence, passive targeting was evidenced by the prolonged plasma circulation and higher tissue accumulations of the conjugates in comparison with free methotrexate. The ratios of drug accumulation at the tumor versus the major site of side effects (small intestine) for both conjugates were enhanced by the EPR effects. The difference in the drug accumulation at the tumor site was insignificant between conjugates with MMP-sensitive and MMP-insensitive linkers. We concluded that the tumor targeting effect of the dextran-peptide-methotrexate conjugate was dominantly due to passive targeting and EPR. The difference in the systemic side effects observed for the conjugates with different linkers was attributed to their varying susceptibility towards enzymes in normal tissues.
by Ying Chau.
Ph.D.

The work represents an initial appraisal ofhow polymer-drug conjugates based upon hyaluronic acid (HA) may behave at sites of inflammation relative to unmodified HA. Three polymer drug conjugates were synthesized in which a model drug, p-nitroaniline (pNA) has been joined to HA via a peptide spacer group containing alanine (ala) and valine (val). The conjugates are: HA-ala-pNA, HA-val-ala-pNA, and HA-ala-ala-ala-pNA. Each of the conjugates was subjected to the degradative effects of both hyaluronidase and hydroxyl radicals by the Fenton reaction (generated by Fe2 + ions and H202) . Hyaluronidase digestion was followed both by measurement of reducing hexosamine end-groups and rheologically. The degradative effects of hydroxyl radicals were followed by rheology alone. On incubation with hyaluronidase, end-group analysis showed that the rate of degradation of HA-ala-ala-ala-pNA preparation did not appear to differ significantly from that of unmodified HA. The hyaluronic acid-val-ala-pNA sample appeared to show a higher degradation rate than HA itself initially, after which it slowed relative to HA itself. With HA-ala-pNA the rate of hydrolysis of the HA backbone was found to be considerably lower that that observed for the other materials. Viscometric studies on the effects of hydroxyl radicals showed that, as expected, the rate of decrease in viscosity of the unmodified HA increased with increasing [Fe2J and that H202 alone showed a depolymerising effect on the HA in a concentration-dependent manner. The initial viscosity of the HA-ala-pNA and HA-ala-ala-ala-pNA was considerably lower than that of the unmodified H.-and hence any degradation was difficult to follow although some effects could Q\? observed with the higher concentrations of Fe::+. The HA-\al-ala-pNA conjugate showed a higher initial viscosity than native HA. The reason for this is not clear and would require more experimentation. Like the unmodified hyaluronic acid, on exposure to hydroxyl radicals, a rapid initial depolymerization of the conjugates was observed the rate of which increased with increasing concentration of Fe:!+.

Thesis (MSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: The combination of natural and synthetic polymers allow for the synthesis of advanced hybrid copolymers. These hybrid copolymers have applications in biomedical areas, one such area being in drug delivery systems (DDS). In this study, a modular approach was used to prepare amphiphilic block copolymers with the ability to self-assemble into three dimensional structures. Reversible addition-fragmentation chain transfer (RAFT) was the synthetic tool used to mediate the polymerization of N-vinylpyrrolidone. RAFT is a versatile method to prepare polymers with control over molecular weight and dispersity. A xanthate chain transfer agent (CTA) was used to obtain the hydrophilic poly(N-vinylpyrrolidone) (PVP) block. An aldehyde functionality could be introduced due to the lability of the xanthate moiety, the procedure of which was effectively optimized to produce quantitative conversion. A dixanthate CTA was synthesized to produce a PVP chain which after the modification reaction, resulted in a α,ω-telechelic polymer. A polypeptide was synthesized via the ring-opening polymerization of Ncarboxyanhydrides (ROP NCA). The living and controllable ROP of NCAs is a method which results in polypeptides, but without a well-defined amino acid order. Poly(γ- benzyl-L-glutamate) (PBLG) was synthesized with a narrow dispersity (Đ = 1.10 – 1.15) using conditions that promote the retention of a terminal primary amine. A protected cysteine functionality was introduced via the terminal amine PBLG chain-end, using peptide synthesis techniques. This resulted in the conjugation of the aldehyde functional PVP and the cysteine terminal PBLG using a covalent, non-reducible thiazolidine linkage. The deprotection of the cysteine, more specifically the deprotection of the thiol was a non-trivial procedure. The thiol protecting acetamidomethyl (Acm) group could not be cleaved using traditional methods, but instead a modified procedure was developed to effectively remove the Acm group while inhibiting hydrolysis of the benzyl esters. It was determined that the conjugation reaction could effectively proceed in N,Ndimethylformamide (DMF) at a slightly elevated temperature and so continued to prepare the amphiphilic hybrid block copolymers, PVP-b-PBLG. A structurally different PBLG chain, namely PBLG-b-Cys was conjugated to the ω-aldehyde PVP and the conjugation efficiency was compared to our PBLG-Cys block. In the case of PBLG-b- Cys the in situ deprotection and conjugation as well as a two-step deprotection and conjugation reaction with PVP resulted in very low conjugation efficiency. The cysteine end-functional PBLG resulted in near quantitative conjugation with PVP. The critical micelle concentration (CMC) for PVP90-b-PBLG54 was determined to be 6 μg/mL, using fluorescence spectroscopy. Particle sizes were determined with TEM and DLS and found to range from 25 nm to 120 nm depending on the polymer block lengths as well as hydrophobic/hydrophilic block length ratios. Furthermore, when the micelles were subjected to an increased acidic environment, the labile benzyl ester bonds were hydrolyzed. This was observed with TEM where the particle sizes increased 10-fold to form vesicular structures. Hydrolysis was further confirmed with ATR-FTIR and 1H-NMR spectroscopy. Cytotoxicity tests confirmed that the copolymer micelles had good cell compatibility at high concentrations such as 0.9 mg/mL. Investigation into drug loading using a pyrene probe confirmed the viability of using PVP-b-PBLG as a responsive DDS.
AFRIKAANSE OPSOMMING: Die kombinasie van natuurlike en sintetiese polimere maak dit moontlik vir die sintese van gevorderde hibried kopolimere. Hierdie kopolimere het aanwending in biomediese gebiede, een so 'n gebied is in medisinale vervoer sisteme (MVS). 'n Modulêre benadering is in hierdie studie gebruik om amfifiliese blok kopolimere te berei. Omkeerbare addisie-fragmentasie kettingoordrag (OAFO) is gebruik as die sintetiese tegniek vir die polimerisasie van N-vinielpirolidoon (NVP). OAFO is 'n veelsydige metode om polimere te berei met beheer oor molekulêre gewig en dispersiteit (Đ). 'n Xantaat kettingoordrag agent (KOA) is gebruik om die hidrofiliese poli(N-vinielpirolidoon) (PVP) blok te sintetiseer. ‘n Aldehied endgroep was deur die terminale xantaat funksionaliteit berei, ‘n proses wat geoptimiseer is tot kwantitatiewe omsetting. 'n Di-xantaat KOA is gesintetiseer om, na modifikasie, 'n α, ω-telecheliese polimeer te produseer. Die polipeptied was gesintetiseer deur middel van ’n ringopening polimerisasie van Nkarboksianhidriede (ROP NKA). Die lewende en beheerbare ROP van NKAe is 'n metode wat lei tot polipeptiede sonder ’n gedefinieerde aminosuur volgorde. Poli(γ- benzyl-L-glutamaat) met 'n lae dispersiteit (Đ = 1.10 – 1.15), is gesintetiseer deur gebruik te maak van kondisies wat die behoud van 'n terminale primêre amien bevorder. 'n Beskermde sistien-funksionaliteit is ingebou via die terminale amien met behulp van peptiedsintese tegnieke. Die tiol beskerming van die asetamidometiel (Asm) groep kon nie gekleef word deur gebruik te maak van tradisionele metodes nie, maar ‘n nuwe proses is ontwikkel om die Asm groep te kleef sowel as om die hidrolise van die bensiel esters te inhibeer. Die koppelings reaksie het effektief verloop in DMF by 'n effens verhoogde temperatuur en sodoende is die amfifiliese hibried blok-kopolimere, PVP-b-PBLG berei. Twee verskillende PBLG kettings is gekoppel aan die ω-aldehied PVP en die koppeling doeltreffendheid is vergelyk. Daar is bevind dat net die sistien end-funksionele PBLG tot kwantitatiewe konjugasie kon lei. Die kritiese misel konsentrasie is bepaal vir PVP90-b-PBLG54 as 6 μg/mL met behulp van fluoressensie spektroskopie. Die deeltjie-groottes is bepaal met TEM en DLS en wissel van 25 nm tot 120 nm, afhangende van die polimeer bloklengtes sowel as hidrofobiese / hidrofiliese blok lengte verhoudings. Die miselle is blootgestel aan 'n verhoogde suur omgewing, wat tot die hidrolise van die bensiel ester groepe gelei het. TEM het getoon dat die deeltjie-groottes met 10-voud vergroot het tot vesikulêre strukture. Hidrolise is verder bevestig met ATR-FTIR en 1H-KMR spektroskopie. Sitotoksiese toetse het bevestig dat die miselle geen of min toksisiteit toon teenoor eukariotiese selle nie, selfs teen 'n hoë konsentrasies soos 0.9 mg/ml. Die medisinale behoud vermoë is met behulp van pireen bevestig en dus ook die potensiaal van PVP-b-PBLG as ‘n moontlike MVS.

Thesis (M.S.)--University of Akron, Dept. of Biomedical Engineering, 2008.
"August, 2008." Title from electronic thesis title page (viewed 12/9/2009) Advisor, Stephanie T. Lopina; Committee members, Amy Milsted, Daniel B. Sheffer, Daniel Ely; Department Chair, Daniel B. Sheffer; Dean of the College, George K. Haritos; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.

The overall aim of my PhD research was to develop biocompatible materials, namely poly(2-oxazoline)s, in terms of chemical structures and chemical and physical properties for drug delivery systems. This thesis demonstrated novel strategies and unique approaches towards sophisticated drug delivery formulations. A combination of crosslinking chemistry, thermoresponsive properties, and drug conjugation was introduced to overcome common issues in typical drug delivery devices such as burst drug release and low drug efficiency. Ultimately, this thesis aimed to promote poly(2-oxazoline)s as the most promising emerging polymers in the future.

Although survival rates of colorectal cancer (CRC) treated with surgery and conventional chemotherapy are high, metastatic CRC still shows acute mortality rates. Current chemotherapeutic treatment involves high doses of cytotoxic drugs, particularly adjuvant combinations of 5-Fluorouracil (5-FU) and Irinotecan (prodrug of SN-38). However, these treatments cause undesirable effects to the patients, which can negatively contribute to their survival. The use of polymer-drug conjugates (PDC) has attracted great attention in the field of controlled drug delivery for cancer treatment, improving the ratio of cytotoxic drugs in tumour tissues, taking advantage of the enhanced permeability retention effect, and consequently reducing the toxicity in healthy tissues. In this thesis the poly-(L-glutamic acid) (PGA) biodegradable polymer has been chosen as the carrier of the designed polymer-drug conjugates. By reason of the chemotherapeutic agent 5-FU is the mainstay of cytotoxic therapy against advanced CRC, in all PDC studied 5-FU has been conjugated to PGA. Three different types of PGA-based PDC have been studied: (i) PGA-5-FU conjugates; (ii) PGA-MMPpept-5FU conjugates using enzymatically-cleavable linkers, particularly MMP-sensitive peptides since some MMPs levels increase as CRC progress; and (iii) PGA-5FU-SN38 conjugates to evaluate the synergism between 5-FU and SN-38 conjugated in a single PDC. Regarding the PGA-5-FU conjugate, it was demonstrated that the conjugation of 5-FU to a PGA carrier through an ester bond showed therapeutic activity in vitro in the HCT-116.Fluc2-C9 and HT-29.FlucC4 CRC cell lines. Cellular uptake experiments performed with PGA-5-FU labelled with carboxyfluoresceine fluorescent probe indicated that the internalization of the PDC was through the endocytic pathway. In addition, in vivo biodistribution experiments of PGA-5-FU labelled with an AlexaFluor dye confirmed that the accumulation in tumor was maximal at 4 h post-adminstration, confirming also that the product was excreted through the kidney, but also removed by the liver in smaller amounts. In the study of a new PDC sensitive to MMP7 to deliver 5-FU we confirmed that it is of significant importance the type of link between the MMP7-sensitive peptide (AHX-RPLALWRS-AHX) and the bioactive agent 5-FU. It was confirmed through cytotoxic experiments of the peptide-drug unit that the carbamate union was too stable in vitro, opposite to the ester bond behavior, resulting as an undesirable linkage for their use in polymeric nanoconjugates designed to deliver drugs gradually. Then, in vitro therapeutic efficacy studies performed in HCT-116.Fluc2-C9 and HT-29.Fluc-C4 cells overexpressing MMP7, showed that the conjugation of 5-FU linked to AHX-RPLALWRS-AHX peptide through an ester bond to a PGA carrier, lead a PDC that adopted a conformation in solution that resulted very accessible for the MMP7 enzyme. When comparing PGA-MMP7pept-5FU with the PGA-5-FU system, it was confirmed that the nature and length of the peptide linker is of significant relevance on the final conformation of the conjugate, therefore on the kinetic release of the drug. Finally, a family of PGA-5FU-SN38 conjugates through an ester bond carrying different proportions of drugs was synthesized using PGA carrier with a molecular weight of 15 KDa. In vitro studies showed an improvement of the cytotoxic activity in the HCT-116.Fluc2-C9 and HT-29.Fluc4 CRC cells of the conjugates carrying both agents in a ratio of SN-38/5-FU of 1:40 and 1:300 in comparison with the behavior observed with the combined drugs as single agents at the same ratio. It was studied the synergy through the calculation of the Combination Index, and we confirmed that the conjugation of SN-38 and 5-FU in a single polymeric vehicle was traduced in a strong synergic interaction between both drugs when compared to the single PDC.
El agente 5-fluorouracilo (5-FU) es el tratamiento quimioterapéutico sistémico esencial para el tratamiento del cáncer colorectal. Sin embargo, la supervivencia global y superación de la enfermedad de pacientes tratados con 5-FU como primera línea de tratamiento es sólo del 10-15%. El uso de conjugados de polímero-fármaco (PDC) ha atraído gran atención en el campo de la administración controlada de fármacos para el tratamiento del cáncer. Éstos mejoran la acumulación de agentes citotóxicos en tejidos tumorales, aprovechando las características en la vascularización de los tumores, por consiguiente reduciendo la toxicidad en los tejidos sanos. Se han estudiado tres tipos diferentes de PDC basados en el polímero biodegradable ácido poly-(L-glutámico)(PGA): (i) Conjugado de PGA-5-FU; (ii) Conjugados de PGA-MMPpept-5FU utilizando enlazadores escindibles enzimáticamente, especialmente péptidos sensibles a MMP-7, ya que los niveles de algunas MMP aumentan a medida que el progresa el CRC; y (iii) conjugados de PGA-5FU-SN38 para evaluar la sinergia entre 5-FU y SN-38 conjugado en un solo vehículo. El conjugado PGA-5-FU mostró actividad terapéutica in vitro en las líneas celulares HCT-116.Fluc2-C9 y HT-29.FlucC4 e internalización mediante endocitosis, mediante la técnica de microscopía confocal. Además, experimentos de biodistribución in vivo confirmaron la acumulación en el tumor y la excreción vía renal y hepática. Con el conjugado PGA-MMP7-5FU se confirmó que la importancia del tipo de enlace entre el péptido MMP7 sensible (AHX-RPLALWRS-AHX) y el agente 5-FU; ya que se observó que la unión carbamato era demasiado estable in vitro, en comparación con la unión éster. Los estudios de citotxicidad in vitro con sobreexpresión de MMP7, confirmaron que la conformación en solución resultó muy accesible para la enzima MMP7. Finalmente, se sintetizó una familia de conjugados PGA-5FU-SN38 con diferentes proporciones de fármacos. Se estudió la sinergia entre ambos mediante el cálculo del índice de combinación, y se confirmó que la conjugación de SN-38 y 5-FU en un solo vehículo polimérico mostraba una fuerte sinérgica entre ambos fármacos en comparación con los PDC cargados con una única droga.

La vectorisation de médicaments a mis en exergue de réelles innovations pharmacologiques et pharmacocinétiques. Néanmoins, peu d’études originales ont été menées sur les vecteurs à visée antiparasitaire. Seuls des médicaments pour lesquels les parasites ont développé des mécanismes de résistance, ont été utilisés en vectorisation. Les conditions et coûts de préparation de ces nanoformulations sont également un frein majeur à leur future production. L’enjeu consistait donc à développer des nanovecteurs permettant la délivrance de nouvelles classes d’antiparasitaires, tout en s’appuyant sur une stratégie de synthèse « click » à la fois accessible et innovante. Dans un premier temps, le potentiel antipaludique de nouveaux sels d’iminium N-hétérocycliques a été évalué sur Plasmodium falciparum, responsable du paludisme. Les sels de bis-aminopyridiniums se sont avérés être les plus prometteurs avec des activités de l’ordre du sub-micromolaire et un mécanisme d’action original probablement lié à leurs forts pouvoirs rédox. Dans un deuxième temps, la vectorisation de ces candidats bis-aminopyridinium a été entreprise par la préparation de nanovecteurs de type conjugués polymère-médicament. Ces derniers ont été obtenus via une stratégie simple consistant en l’utilisation du bis-aminopyridinium comme initiateur de la polymérisation de son propre nanovecteur. L’étude des RSA a souligné l’importance d’un premier bloc acrylate et d’un arrangement sphérique sur l’activité. Ainsi, un système tribloc soluble, biodégradable et furtif, de tailles inferieures à 100 nm, vectorisant 5 mol% de principe actif, a permis le maintien d’une bonne activité antiplasmodiale, sans effet cytotoxique
Drug delivery has emphasised real pharmacological and pharmacokinetic advancements. Nevertheless, few in-depth and original studies have been conducted on antimalarial vectors. Only approved drugs, for which the parasites have developed resistance mechanisms, have been used in vectorization. The conditions and costs of preparation of these nanoformulations constitute a major barrier to their future production. This project aimed at developing nanovectors for the delivery of new antiparasitic drugs, while relying on a both accessible and innovative "click" synthesis strategy. First, the antimalarial efficiency of new N-heterocyclic iminium salts was evaluated on Plasmodium falciparum, responsible for malaria. Bis-aminopyridinium salts proved to be the most promising candidates with sub-micromolar antiplasmodial activities and an original mechanism of action, probably related to their strong redox properties. Secondly, the vectorization of these bis-aminopyridinium drug candidates was undertaken through preparation of polymer-drug conjugate nanovectors. The latter were obtained via a simple strategy, consisting in the use of the bis-aminopyridinium salt as polymerization initiator of its own nanovector. The SAR study underlined the need for a first acrylate block and spherical arrangements on the activity. Hence, a soluble, biodegradable, and stealth triblock system with sizes lower than 100 nm and enabling the delivery of 5 mol% of drug, showed effective antiplasmodial activity, without any cytotoxic effect

Oxidative stress, the imbalance of free radical generation with antioxidant defenses, leads to cellular inflammation, apoptosis and cell death. This compromised environment results in debilitating diseases, such as oral mucositis (OM), atherosclerosis, and ischemia/reperfusion injury. Antioxidant therapeutics has been a proposed strategy to ameliorate these imbalances and maintain homeostatic environments. However, the success of these approaches, specifically curcumin, has been limited due to characteristics such as hydrophobicity and high reactivity when released as bolus doses to contest to oxidative stress induced diseases. The development of a controlled release system to aid in protection of the antioxidant capacity of curcumin, as well as a tunable system to aid in proper rate of release for disease can overcome these limitations. Previously, the use of a poly(beta-amino ester) (PBAE) chemistry has been developed in Dziubla and Hilt laboratories to provide desirable properties. The dynamic mechanical analysis and efficacy in cellular protection has been studied, yet the sensitivity and responsiveness of these polymers to abnormal environments found within oxidative stress compromised environments are unknown. In this work, a series of networks were comprised of different molar ratios of modified acrylated curcumin, poly(ethylene glycol) diacrylate, and a primary diamine crosslinker to create tunable hydrolytically degradable crosslinked hydrogels. I hypothesized a consumption rate difference of free curcumin and curcumin as a released product from the crosslinked network in the presence of a free radical generating system. After the consumption profiles of each were reported differently, the experimental data was translated into a kinetic rate model to identify quantitative consumption rate parameters of curcumin and active film degradation products. The effect on the released products arose the question of curcumin consumption in other oxidizing environments. These networks were then investigated in low concentrations of a hydrogen peroxide insult, and interestingly showed sensitivity to hydrolysis by recovering significantly more curcumin at an accelerated rate of release. Identifying the sensitivity of these tunable networks to environmental stimuli, they were then presented to a series of low pH environments, which significantly reduced the degradation time, finding a dependence of rate of release on the weight loading of curcumin present within the film. To translate these responsive materials to an application-based system, the curcumin conjugated PBAE polymers were investigated as an oral rinse drug delivery system for the treatment of radiation-induced OM in a hamster model. Radiation-induced OM onset and severity was reduced with a 20 wt% microparticle loaded mucoadhesive system that releases curcumin over 24 hours, providing promising results of a therapeutic effect from curcumin when incorporated in to a controlled release delivery system. Overall, curcumin conjugated PBAE polymers show selectivity of hydrolysis in abnormal environments related to oxidative stress. This information is beneficial to the proper design and loading of antioxidant therapeutics within crosslinked polymers, giving the ability to tune release to treat and deliver based on the environment’s insult. This can advance the potential use for antioxidant therapeutics in pharmaceutical applications in the future.

碩士
國立中山大學
化學系研究所
107
Recently, biological medicine has gradually replaced the status of traditional chemical medicine in global pharmaceutical market. Among them, ADCs (Antibody-drug conjugate) has high site-specific for tumor cell and low systemic toxicity compared with traditional chemotherapy drugs, providing a new selection for future cancer treatment. In this research, we use our published boronic acid (BA)-tosyl initiator probe to have glycan-directed modification of IgG, and grow PEG from initiator site by Atom-transfer radical-polymerization (ATRP). After polymerization, we use 5% KOH to cut PEG from IgG, and then analyze polymer length by GPC spectrometer. To construct a initial ADC backbone model, we try to find out the correlation with reaction conditions and PEG length by GPC data. And we could also find the difference of IgG heavy chain by SDS-PAGE, before and after using KOH solution. This result prove again our probe has specific modification of IgG Fc domain. Lastly, we attempted to construct a preliminary of Antibody-polymer drug conjugate model. We replace cytotoxin by p-nitroaniline, and attach it to Val-Ala-PBAC dipeptide linker. The concentration of p-nitroaniline could be detected by UV absorption spectrometer, and then grafting efficiency would be estimated. Hoping that grafting efficiency could provide a preliminary standard for further cytotoxic ADC drug grafting efficiency. And then quantified the number of drugs which grafted on antibody, we wish to provide a more accurate value for the future clinical drug delivery.

Nanoparticle (NP) drug delivery is a major focus in the research community because of its potential to use existing drugs in safer and more effective ways. Chemotherapy encapsulation in NPs shields the drug from the rest of the body while it is within the NP, with less systemic exposure leading to fewer off-target effects of the drug. However, passive loading of drugs into NPs is a suboptimal method, often leading to burst release upon administration. This work explores the impact of incorporating the drug-polymer conjugate doxorubicin-poly (lactic-co-glycolic) acid (Dox-PLGA) into a lipid-polymer hybrid nanoparticle (LPN). The primary difference in using a drug-polymer conjugate for NP drug delivery is the drug’s release kinetics. Dox-PLGA LPNs showed a more sustained and prolonged release profile over 28 days compared to LPNs with passively loaded, unconjugated doxorubicin. This sustained release translates to cytotoxicity; when systemic circulation was simulated using dialysis, Dox-PLGA LPNs retained their cytotoxicity at a higher level than the passively loaded LPNs. The in vivo implication of preserving cytotoxic potency through a slower release profile is that the majority of Dox delivered via Dox-PLGA LPNs will be kept within the LPN until it reaches the tumor. This will result in fewer systemic side effects and more effective treatments given the higher drug concentration at the tumor site. An intriguing clinical application of this drug delivery approach lies in using Dox- PLGA LPNs to cross the blood-brain barrier (BBB). The incorporation of Dox-PLGA is hypothesized to have a protective effect on the BBB as its slow release profile will prevent drug from harming the BBB. Using induced pluripotent stem cells differentiated to human brain microvascular endothelial cells that comprise the BBB, the Dox-PLGA LPNs were shown to be less destructive to the BBB than their passively loaded counterparts. Dox-PLGA LPNs showed superior cytotoxicity against plated tumor cells than the passively loaded Dox LPNs after passing through an in vitro transwell BBB model. Dox-PLGA LPNs and drug-polymer conjugates are exciting alternatives to passively loaded NPs and show strong clinical promise of a treatment that is more potent with fewer side effects and less frequent administration.

Student Number : 9903022H MSc Dissertation School of Chemistry Faculty of Science
Cancer is characterised by the unconstrained growth of cancerous cells, which damages the healthy cells and ultimately the tissue of the host. Chemotherapy forms an essential component in the treatment of this disease, however most anti-tumour drugs suffer from various deficiencies, e.g. increased toxicity, reduced serum half life and poor water solubility. The focus of this project was to address some of these deficiencies by conjugating selected drugs to a water-soluble polymeric carrier. Selected water-soluble biodegradable carriers were synthesized. Copolyaspartamides, polyamidoamines and polyamides were obtained by condensation polymerisation, Michéal–type addition polymerisation and ester amine base-catalysed polymerisation. The nascent water soluble polymers were used to conjugate platinum, ferrocene and tetramethylmelamine derivative, respectively. The percentage drug in each polymer drug conjugate was determined by considering the mass of the drug in the conjugate as a percentage of the total mass of the drug-polymer conjugate. Platinum was linked to the carrier via polymer attached amine, carboxyl and hydroxyl ligands. Platinum content of the conjugates ranged from 7 to 11 % by mass. The ferrocenylation agent, 4-ferrocenylbutanoic acid, and the tetramethylmelamine derivative, 3-(4,6-bis(N,N-dimethylamino)-1,3,5-triazacyclohexatrien-2-yl) propanoic acid was polymer-bound by amidation reactions. Iron content of the ferrocence conjugated ranged from 2 to 12 % by mass. While the drug content based on tetramethylmelamine in the 3-(4,6-bis[N,N-dimethylamino]-1,3,5-triazacyclohexatrien-2-yl) propanoic acid polymer conjugate ranged from 8.4 to 8.6 % by mass. There was a preliminary attempt to coconjugate both, 4-ferrocenylbutanoic acid and 3-(4,6-bis[N,N-dimethylamino]-1,3,5- triazacyclohexatrien-2-yl) propanoic acid to the same polymer. This co-conjugate contained 2.9 % iron and 3.4 % tetramethylmelamine by mass.

Polymer networks are promising biomaterials for drug delivery as they have porous structures and are often biocompatible. The general aspects of the host-guest complexation capability of polymer networks containing cyclodextrins as well as their application in drug delivery are considered in Chapter 1. The introduction of cyclodextrins into polymer networks has the potential to improve drug loading capacity and modulate subsequent drug release behavior due to the host-guest complexation by cyclodextrins of drug molecules. Thus, Chapter 2 and Chapter 3 are concerned with new research on water soluble β-cyclodextrin, adamantyl and octadecyl substituted poly(acrylate) networks, respectively, as potential sustained drug delivery systems. In Chapter 2, research into self-assembled poly(acrylate) networks cross-linked through host-guest complexation between β-cyclodextrin, β-CD, substituents and adamantyl, AD, substituents as potential sustained drug delivery systems is described. A poly(acrylate) (PAA) 8.8% randomly substituted with β-CD through an ethyl tether, PAAβ-CDen, is synthesized as a host poly(acrylate). Poly(acrylate)s 3.3%, 3.0% and 2.9% randomly substituted with AD substituents, respectively, through ethyl, hexyl and dodecyl tethers in the PAAADen, PAAADhn and PAAADddn are synthesized as guest polymers. The host-guest complexation of PAAADen, PAAADhn, and PAAADddn by PAAβ-CDen in aqueous solution produce three self-assembled poly(acrylate) networks. These complexations are characterized by isothermal titration calorimetry, ITC, 2D NOESY ¹H NMR spectroscopy, and rheology. It is found that the length of the tether between the AD group and the poly(acrylate) backbone has a substantial influence on the complexation constants, KITC [ITC subscript], as well as the associated enthalpy change, ΔH, and entropy change, ΔS. The smallest and largest KITC [ITC subscript] occur for PAAADen with the shortest tether and PAAADddn with the longest tether, which coincides with the lowest and highest viscosities occurring for the aqueous PAAβ-CDen/PAAADen and PAAβ-CDen/PAAADddn networks. The complexation of three different dye molecules, acting as drug models, by the β-CD substituents in these networks is characterized by UV-Vis spectroscopy and 2D NOESY ¹H NMR studies. The results suggest that dye complexation by the β-CD substituents in the three poly(acrylate) networks is weaker by comparison with the complexation by native β-CD and PAAβ-CDen, as indicated by decreased complexation constants. The poly(acrylate) networks exhibit complexation-controlled dye release behavior, and thereby sustained dye release profiles. Thus, the three poly(acrylate) networks studied, which form hydrogels at higher concentrations, have substantial potential as sustained drug delivery systems. In Chapter 3, the complexation and release behavior of six dyes in a β-CD- and octadecyl-substituted poly(acrylate) network is explored to further extend the understanding of the host-guest complexation between β-CD substituents and guest molecules within a fourth polymer network system and its influence on the release of guest molecules from the polymer network. Thus, β-CD substituents are 9.3% randomly substituted onto poly(acrylate) through an ethyl tether to give PAAβ-CDen and octadecyl, C18, substituents are 3.5% randomly substituted onto poly(acrylate) to give PAAC18. The network forms through the host-guest complexation between the β-CD substituents and C18 substituents, and is characterized by a complexation constant of K = 1.13 × 10⁴ dm³ mol⁻¹, associated with ΔH = −21.55 kJ mol⁻¹ and TΔS = 1.59 kJ mol⁻¹. The complexation of the dyes by the β-CD substituents in the PAAβ-CDen/PAAC18 network is characterized by UV-Vis absorption and fluorescence spectroscopy and 2D NOESY ¹H NMR studies. The results suggest that the complexation of dyes by the β-CD substituents in the PAAβ-CDen/PAAC18 network is weaker by comparison with the complexation by native β-CD and PAAβ-CDen, as indicated by decreased complexation constants. The PAAβ-CDen/PAAC18 network exhibits complexation-controlled dye release behavior and thereby sustained dye release profiles. Thus, the PAAβ-CDen/PAAC18 network, or hydrogel at higher concentration, is a potential sustained drug delivery system. Conjugated polymer nanoparticles are promising fluorescent probes as a consequence of their high brightness and photostability. Chapter 1 introduces the general methods of preparing conjugated polymer nanoparticles and their wide ranges of biological applications. However, conjugated polymer nanoparticles exhibit large-scale aggregation and precipitation at the high ionic strengths encountered under physiological conditions, which presents an impediment to their biological applications. In seeking to address this issue, the research described in Chapter 4 and Chapter 5 addresses stabilization of conjugated polymer nanoparticles using hydrophobic linear alkyl group substituted poly(acrylate)s and bovine serum albumin and explores their deployment in cell imaging applications. In Chapter 4, the synthesis of hydrophobic linear alkyl group substituted poly(acrylate)s, PAACn, is described as is their employment as conjugated polymer nanoparticle stabilizers. (When n = 18, 16 and 10 the alkyl groups are octadecyl, hexadecyl and decyl, respectively.) The carboxylate groups of PAACn increase the surface charge of the conjugated polymer nanoparticles and thereby stabilize them in phosphate buffered saline, PBS. Nanoparticles of the green-yellow emitting conjugated polymer, F8BT, stabilized with PAACn, F8BT-PAACn, are prepared using a nano-precipitation method. In contrast to the significant aggregation with a negligible yield (~ 0%) of bare F8BT nanoparticles in PBS, high yields approaching 90% are observed for F8BT nanoparticles stabilized with PAAC18 at 1%, PAAC16 at 3%, and PAAC10 at 10% substitution. The F8BT-PAACn nanoparticles have small sizes ranging from 50 to 70 nm in diameter, highly negative surface charge and high colloidal stability over 4 weeks in PBS. These properties pave the way for the deployment of F8BT-PAACn nanoparticles in biological applications. Spectroscopic results indicate the PAACn has no adverse effect on the UV-Vis absorptivity and fluorescence brightness of F8BT-PAACn nanoparticles relative to bare F8BT nanoparticles. In addition, F8BT-PAACn nanoparticles are internalized by HEK 293 cells and exhibit negligible cytotoxicity. Thus, PAACn are versatile and robust stabilizing materials that facilitate the application of F8BT-PAACn nanoparticles as fluorescent probes in cell imaging.The research described in Chapter 5 shows that bovine serum albumin, BSA, stabilizes conjugated polymer nanoparticles in phosphate buffered saline, PBS, evidently due to the combined effects of the negatively charged surfaces arising from the BSA carboxylate groups and the steric effect of the bulk 3D structure of BSA. Three multicolored conjugate polymers, PDOF, F8BT, and MEHPPV, are employed to prepare their corresponding nanoparticles using a nano-precipitation method. In contrast to the significant aggregation with negligible yields (~ 0%) of bare conjugated polymer nanoparticles occurring in PBS, high yields approaching 100% are observed for conjugated polymer nanoparticles stabilized with BSA, CP-BSA nanoparticles, in PBS. These CP-BSA nanoparticles have small sizes ranging from 20 to 60 nm, negative surface charges and high colloidal stability. Spectroscopic results indicate the BSA has no adverse effect on the UV-Vis absorptivity and fluorescence brightness of the CP-BSA nanoparticles relative to bare conjugated polymer nanoparticles. These properties potentially pave the way for the deployment of these CP-BSA nanoparticles in biological applications.
Thesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 2016.