Abstract Macrophages found in the tumor microenvironment play a crucial role in initiating an immunosuppressive tumor microenvironment that negatively impacts immunotherapy efficacy and aids tumor progression and metastasis. Constituting the most abundant immune cell in tumor microenvironment (TME), tumor associated macrophages (TAMs) have emerged as an attractive approach for anti-cancer therapy. However, two major challenges need to be overcome for successfully utilizing macrophages for immunotherapy. First, tumors repolarize the TAMs predominantly to M2 tumor-aiding phenotype by secreting various immunosuppressive cytokines. Second, cancer cells overexpress a membrane protein CD47 that interacts with signal-regulating protein alpha (SIRPalpha) expressed on macrophages. This crosstalk provides a downregulatory signal in the form of activation of SHP1/2 that inhibits cancer cell phagocytosis, and CD47, therefore, functions as a “don’t-eat-me” signal. We rationalized that these challenges can be overcome by engineering a nanoparticle system that can deliver a rationale combination of immunomodulatory agents to the TAMs that can both repolarize the M2 macrophages to M1 phenotype efficiently and concurrently block CD47-SIRPalpha interactions by inhibiting SHP2 signaling. Herein, we designed a lipid nanoparticle (LNP) system loaded with amphiphilic R848-cholesterol in its hydrophobic lipid bilayer, while SHP099 gets encapsulated in the hydrophilic core. Our previous studies have shown that the conjugation of cholesterol to the inhibitor stabilizes the lipid bilayer at a high inhibitor concentration. The LNPs showed high optimal drug loading, size, and stability. In vitro studies showed that the M2 macrophages treated with the LNPs system repolarized to M1 phenotype and expressed co-stimulatory molecules while having enhanced phagocytic potential. In vivo efficacy studies in 4T1 tumor-bearing mice showed that LNPs exhibit superior anti-tumor efficacy compared to other treatments. We evaluated the effect of MARCO-targeted LPNs by the conjugating anti-MARCO antibody on the LPN surface. However, no comparable difference in treatment efficacy was observed between the targeted MARCO-LNPs and the non-targeted LNPs. These results demonstrate that the MARCO targeting system designed in this study is largely ineffective in the targeted delivery of its drug cargo specifically to TAMs. Thus, the lipid nanoparticle-mediated co-delivery of a rational combination of TLR7/8 agonist and SHP2 inhibitor in the TAMs increases M2 to M1 repolarization and phagocytosis potential of macrophages. Recommended Citation Malik, V., Ramesh, A. and Kulkarni, A.A. (2021), TLR7/8 Agonist and SHP2 Inhibitor Loaded Nanoparticle Enhances Macrophage Immunotherapy Efficacy. Adv. Therap., 4: 2100086. https://doi.org/10.1002/adtp.202100086

No description available.

A range of microgels with two different functionalities were synthesized, and their utility in novel bioapplications was examined. Cationic microgels with varying properties were developed by tuning synthesis conditions. Their size and primary amine content was analyzed, and one microgel system was selected as a model construct. Its primary amine groups were conjugated to two dyes with properties favorable for utilization as contrast agents in photoacoustic imaging. The concentration of contrast agent in single particles was determined. The implications of a high local dye concentration in the generation of high intensity photoacoustic signals, are discussed. The second bioapplication involved the targeted delivery of fibrinolytics to fibrin clots, in order to bring about dissolution of abnormal thrombi. For this purpose, core/shell microgels with carboxylic acid groups in their shells were synthesized in three size ranges. Following this, their dimension based differential localization in and around porous fibrin clots was examined. Fibrin-specific peptides were then conjugated onto the shells of these particles and the conjugates were shown to demonstrate strong interactions with the fibrin clots. The microgels conjugated to the peptide with the highest binding affinity to fibrin, were observed to bring about disruption of fibrin clots, merely through interference in the dynamic interactions among clot fibers, due to the equilibrium nature of the fibrin polymer. The implications of these novel results and future studies required to facilitate a better understanding of the phenomena involved, are discussed.

Dans les dernières 15 ans, la recherche biomédicale a exploré en profondeur l’utilisation de nanoparticules pour la délivrance ciblée de médicaments. Parmi plusieurs matériaux étudiés, la silice mésoporeuse représente une plateforme exceptionnelle pour ce type d’applications puisque elle est biocompatible et capable d’être chargé avec une quantité élevée de médicament, tout en étant facile à synthétiser et à fonctionnaliser. La connaissance des interactions entre nanoparticules de silice et environnement biologique est nécessaire pour concevoir des vecteurs thérapeutiques efficaces et pas toxiques. Cet étude a développé une nouvelle méthode d’analyse in situ pour suivre les interactions entre silice mésoporeuse et fluides biologiques réels (sérum et sang), employant une cellule d’analyse microfluidique et l’ellipsométrie en réflexion totale interne. Nous avons ainsi réalisé le suivi dynamique de la dégradation de vecteurs models à base de silice poreuse structuré dans une solution tampon à pH physiologique et une solution concentré de protéines. Ces analyses ont permis d’évaluer l’influence de la structure poreuse, de l’adsorption de protéines sur la surface et de la vitesse du flux sur la dissolution de la silice mésoporeuse. / Dans les dernières 15 ans, la recherche biomédicale a exploré en profondeur l’utilisation de nanoparticules pour la délivrance ciblée de médicaments. Parmi plusieurs matériaux étudiés, la silice mesoporeuse représente une plateforme exceptionnelle pour ce type d’applications puisque elle est biocompatible et capable d’être chargé avec une quantité élevée de médicament, tout en étant facile à synthétiser et à fonctionnaliser .La connaissance des interactions entre nanoparticules de silice et environnement biologique est nécessaire pour concevoir des vecteurs thérapeutiques efficaces et pas toxiques. Cet étude a développé une nouvelle méthode d’analyse in situ pour suivre les interactions entre silice mesoporeuse et fluides biologiques réels (serum et sang), employant une cellule d’analyse microfluidique et l’ellipsometrie en réflexion totale interne. Nous avons ainsi réalisé le suivi dynamique de la dégradation de vecteurs models à base de silice poreuse structuré dans une solution tampon à pH physiologique et une solution concentré de protéines. Ces analyses ont permis d’évaluer l’influence de la structure poreuse, de l’adsorption de protéines sur la surface et de la vitesse du flux sur la dissolution de la silice mesoporeuse.

Submitted by Neusa Fagundes (neusa.fagundes@unioeste.br) on 2018-03-06T19:28:50Z No. of bitstreams: 2 Giovane_Zanin2014.pdf: 3169659 bytes, checksum: ca4852dea2558da8d3c74260c79fc413 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-03-06T19:28:50Z (GMT). No. of bitstreams: 2 Giovane_Zanin2014.pdf: 3169659 bytes, checksum: ca4852dea2558da8d3c74260c79fc413 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-09-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The polyvinyl acetate is a polymer used in the development of formulations for sustained release of drugs. Hyaluronic acid (HA) can interact with receptors on the plasma membrane, especially CD44 that overexpressed in tumor cells. This paper presents a pre-formulation study of isolated polyvinyl acetate films with the addition of HA to the application perspective in pharmaceutical dosage forms destined to modified drug release. The films were prepared using the solvent evaporation method and evaluated according to macroscopic and morphologic characteristics, thickness, Fourier transform infrared spectroscopy (FTIR), thermal analyses (thermogravimetry and differential scanning calorimetry), scanning electron microscopy, water vapor transmission, and swelling index. Addition of HA enabled the formation of isolated films, influencing transparency, flexibility, and thickness. FTIR spectra demonstrated that only physical mixing occurred. TG and DSC curves indicated that films are thermally stable up to 200C. Electron micrographs showed modifications in the polymer mesh structure in samples (85:15 and 80:20) previously immersed in simulated gastric fluid and more pronounced after immersion in simulated intestinal fluid. The HA concentration also influenced water vapor permeability and swelling increasing these indices. We propose that films with 95:05 and 90:10 compositions can be used for modified drug release, as they were similar to a reference film and maintained targeted delivery. / O acetato polivinílico (AcPV) é um polímero utilizado no desenvolvimento de formulações para liberação sustentadas de fármacos. O ácido hialurônico (AH) possui a habilidade de interagir com receptores nas membranas plasmáticas celulares, em especial o CD44 que esta super-expresso em células tumorais. O objetivo deste trabalho foi realizar um estudo de pré-formulação em filmes isolados de acetato polivinílico (AcPV) adicionados de AH na perspectiva de aplicação em formas farmacêuticas destinadas à liberação modificada de fármacos. Os filmes foram produzidos pelo método de evaporação do solvente e avaliados quanto as características macroscópicas e morfológicas, espessura, espectroscopia no infravermelho (FT-IR), análises térmicas (TG e DSC), microscopia eletrônica de varredura, transmissão de vapor de água e índice de intumescimento. Os resultados demonstraram que adição de AH permitiu a formação de filmes isolados adequados influenciando na transparência, flexibilidade e espessura. Espectro no FT-IR evidenciaram ocorrência apenas de mistura física entre os constituintes. As curvas TG e DSC indicaram que os filmes são termicamente estáveis até a temperatura de 200 ºC. Nas micrografias eletrônicas foi possível observar alterações na estrutural da malha polimérica nas composições 85:15 e 80:20 para as amostras previamente imersas em fluido de simulação gástrica, sendo ainda mais pronunciado após imersão em fluído de simulação intestinal. A concentração do AH também influenciou diretamente permeabilidade ao vapor de água e o intumescimento, aumentando estes índices. Na perspectiva de aplicação dos filmes avaliados, destacamos as composições 95:05 e 90:10, as quais sugerem maior potencial para aplicação em formas farmacêuticas para liberação modificada de fármacos uma vez que foram os mais semelhantes ao filme padrão e que mantiveram a possível capacidade de sítio alvo especificidade.

Prostate cancer is one of the most abundant types of cancer among men and the demand for a specific treatment is very high. In this thesis, I have focused on using Glutamate Carboxypepti- dase II (GCPII), as a target for a proof-of-principle delivery system. GCPII is a transmembrane protein that internalizes after a binding of a ligand and is overexpressed in prostate cancer. Virus-like particles from Murine polyomavirus (VLPs) are a suitable nanocarrier for the delivery of imaging agents and drugs. Here I describe modifying these VLPs with inhibitors of GCPII and fluorescent dyes and characterize their binding to GCPII on surface plasmon resonance and to cells expressing GCPII on confocal microscopy. VLPs carrying a GCPII inhibitor show specific binding to GCPII on surface plasmon reso- nance, however they bind non-specifically to cells that don't express GCPII. Several approaches have been tried to avoid that. The substitution of BC loop on the exterior surface of VLPs that is partially responsible for the binding of sialic acid did not seem to affect specificity on cells. Another approach tested was coating of the wild-type VLPs with large polymer carrying a flu- orescent label and a GCPII inhibitor. After the conjugation of the polymer to the VLP, specific binding and internalization in GCPII-positive...

The goal of cancer chemotherapy is to develop effective, safe, and well-tolerated medications. The over-expression of certain receptors on cancer cell membrane provides a basis for active targeting by not only specific interaction between drug delivery system and cells, but also facilitated cellular uptake via receptor-mediated endocytosis. In this study, LDV oligomers up to six LDV repeating units were synthesized via solid phase peptide synthesis method, and evaluated as drug carrier as well as targeting moiety to deliver doxorubicin (Dox) to human malignant melanoma cells (A375), which over-express integrin α 4 β 1 . Cells expressing different levels of integrin α 4 β 1 or modulated using integrin α 4 -specific siRNA knock-down technique were verified by western blot and PCR. Magnetic beads with tripeptides LDV, VDL, or LNV on the surface were used in the binding specificity studies. Results verified that LDV was the minimally required ligand sequence for the specific binding to integrin α 4 β 1 , of which the interaction depends on the amount of integrin and can be utilized for the design of targeted drug delivery. The studies on A375 cells uptake of FITC-labeled LDV oligomers examined the effects of EDTA, temperature, endocytosis inhibitor, and competitive ligand. Cellular uptake mechanism was revealed to be temperature-dependent, receptor-mediated endocytosis, involving the specific interaction between LDV and integrin α 4 β 1 . The internalization extent of LDV monomer was the highest and was also inhibited to the most by the addition of free LDV when compared to other LDV oligomers. Cytotoxicity profiles of Dox-conjugated LDV oligomers were obtained on wild-type A375, integrin α4 knock-down A375, and normal human epithelial keratinocytes (NHEK) using SRB assay. A significant decrease (3∼6 folds) in the cytotoxicity of oligo(LDV)-Dox on A375 cells were observed when the integrin α4 expression was knocked down by ∼50%. Cytotoxicity further decreased on NHEK, which has the lowest integrin α4 expression among three cell lines. In contrast to oligo(LDV)-Dox, free Dox was not able to differentiate between cancerous and normal cells. This result demonstrated the potential of oligo(LDV) as targeting ligand. However, increase of repeating LDV unit did not lead to any apparent trend in cytotoxicity capacity. To facilitate the intracellular Dox release, hydrazone bond (HYD) was introduced between LDV and Dox. In vitro Dox release profiles in pH 6.0, 7.4, and rat plasma proved the pH-sensitivity of LDV-HYD-Dox. Cytotoxicity studies showed an increased cytotoxic effect of LDV-HYD-Dox when compared with LDV-Dox on wild-type A375 (2.5 times), knock-down A375 (1.5 times); while no significant difference in cytotoxicity on NHEK was observed. In vivo animal study supported the in vitro findings on LDV-HYD-Dox, which showed a significant inhibition of tumor growth and longest mice life span when compared to free Dox, poly(L,D,V)-Dox, and LDV-Dox, with averagely only ¼ of the tumor size and almost twice the life span of that from the free Dox group. In conclusion, based on the concept of specific interaction between LDV and integrin α 4 β 1 , oligo(LDV)-Dox targeted drug delivery system was developed and proved to be effective in the delivery of Dox to melanoma cells.

No description available.

Delivery of siRNAs for the treatment of tumors critically depends on the development of efficient nucleic acid carrier systems. The complexation of dendritic polymers (dendrimers) results in nanoparticles, called dendriplexes, that protect siRNA from degradation and mediate non-specific cellular uptake of siRNA. However, large siRNA doses are required for in vivo use due to accumulation of the nanoparticles in sinks such as the lung, liver, and spleen. This suggests the exploration of targeted nanoparticles for enhancing tumor cell specificity and achieving higher siRNA levels in tumors. In this work, we report on the targeted delivery of a therapeutic siRNA specific for BIRC5/Survivin in vitro and in vivo to tumor cells expressing the surface marker prostate stem cell antigen (PSCA). For this, polyplexes consisting of single-chain antibody fragments specific for PSCA conjugated to siRNA/maltose-modified poly(propylene imine) dendriplexes were used. These polyplexes were endocytosed by PSCA-positive 293TPSCA/ffLuc and PC3PSCA cells and caused knockdown of reporter gene firefly luciferase and Survivin expression, respectively. In a therapeutic study in PC3PSCA xenograft-bearing mice, significant anti-tumor effects were observed upon systemic administration of the targeted polyplexes. This indicates superior anti-tumor efficacy when employing targeted delivery of Survivin-specific siRNA, based on the additive effects of siRNA-mediated Survivin knockdown in combination with scFv-mediated PSCA inhibition. Among non-viral vectors, cationic polymers, such as poly(propylene imine) (PPI), play also a prominent role in plasmid DNA delivery. However, limitations of polycationic polymer-based DNA delivery systems are (i) insufficient target specificity, (ii) unsatisfactory transgene expression, and (iii) undesired transfer of therapeutic DNA into non-target cells. We developed single-chain antibody fragment (scFv)-directed hybrid polyplexes for targeted gene therapy of prostate stem cell antigen (PSCA)-positive tumors. Besides mono-biotinylated PSCA-specific single-chain antibodies (scFv(AM1-P-BAP)) conjugated to neutravidin, the hybrid polyplexes comprise β cyclodextrin-modified PPI as well as biotin/maltose-modified PPI as carriers for minicircle DNAs encoding for Sleeping Beauty transposase and a transposon encoding the gene of interest. The PSCA-specific hybrid polyplexes efficiently delivered a GFP gene in PSCA-positive tumor cells, whereas control hybrid polyplexes showed low gene transfer efficiency. In an experimental gene therapy approach, targeted transposition of a codon-optimized p53 into p53 deficient HCT116p53-/-/PSCA cells demonstrated decreased clonogenic survival when compared to mock controls. Noteworthily, p53 transposition in PTEN-deficient H4PSCA glioma cells caused nearly complete loss of clonogenic survival. These results demonstrate the feasibility of combining tumor-targeting hybrid polyplexes and Sleeping Beauty gene transposition, which, due to the modular design, can be extended to other target genes and tumor entities.

Le cancer est la principale cause de mortalité au Canada. Les taxanes (e.g. le paclitaxel et le docétaxel (DCTX)) constituent des remèdes efficaces contre une série de tumeurs solides telles que les cancers du sein, du poumon et de l’ovaire. Par ailleurs, des acides nucléiques (e.g. les oligonucléotides antisens (AON) ou les petits ARN interférents (siRNAs)), capables de supprimer sélectivement certains oncogènes impliqués dans la carcinogénèse, sont actuellement étudiés pour traiter une large gamme de cancers. Bien que l’activité des taxanes et des acides nucléiques soit bien établie sur des modèles humains et/ou animaux, plusieurs aspects physico-chimiques et cliniques restent encore à améliorer. Leur solubilité limitée (pour les taxanes), leur dégradation rapide dans le sang (pour les acides nucléiques), leur élimination précoce, leur absence de sélectivité et leur toxicité envers les tissus sains sont les principaux facteurs limitant leur efficacité. C’est pourquoi de nombreux efforts ont porté sur l’élaboration de systèmes de vectorisation ciblés à base de polymères, dans le but de surmonter les problèmes associés aux thérapies actuelles. Dans cette thèse, deux types de micelles polymères ont été développés pour la vectorisation de DCTX et d’acides nucléiques. D’une part, des micelles de poly(oxyde d’éthylène)-bloc-poly(oxyde de butylène/styrène) ont été étudiées pour la première fois pour solubiliser le DCTX et le protéger de l’hydrolyse. Ces polymères se sont révélés moins toxiques que le surfactant utilisé commercialement pour solubiliser le DCTX (i.e. polysorbate 80) et ont permis une libération prolongée du principe actif. D’autre part, deux systèmes différents de micelles polyioniques (PICM) ont été mis au point pour la vectorisation d’acides nucléiques. De nouveaux conjugués de poly(éthylène glycol) (PEG)-oligonucléotide ont été proposés pour la protection et la libération contrôlée d’AON. Lorsque ces conjugués ont été formulés avec des dendrimères de poly(amidoamine) (PAMAM), des complexes de taille homogène ont été obtenus. Ces PICM ont permis de prolonger la libération de l’AON et de le protéger efficacement contre la dégradation enzymatique. De plus, des polymères de poly(oxyde d’éthylène)-bloc-poly(méthacrylate de propyle-co-acide méthacrylique) ont été incorporés afin de conférer des propriétés acido-sensibles aux PICM. Dans ces micelles, formées de ce dernier polymère formulé avec le dendrimère PAMAM, des oligonucléotides (AON et siRNA) ciblant l’oncogène Bcl-2 ont été encapsulés. L’internalisation cellulaire fut assurée par un fragment d’anticorps monoclonal (Fab’) situé à l’extrémité de la couronne de PEG. Après l’internalisation cellulaire et la protonation des unités d’acide méthacrylique sous l’effet de l’acidification des endosomes, les micelles se sont affranchies de leur couronne. Elles ont ainsi exposé leur cœur composé d’acide nucléique et de dendrimère PAMAM, qui possède une charge positive et des propriétés endosomolytiques. En effet, ces PICM acido-sensibles ciblées ont permis d’augmenter la biodisponibilité des acides nucléiques vectorisés et se sont avérées plus efficaces pour silencer l’oncoprotéine Bcl-2 que les micelles non ciblées ou que le dendrimère de PAMAM commercial seul. Finalement, les nanovecteurs polymères présentés dans cette thèse se révèlent être des systèmes prometteurs pour la vectorisation des anticancéreux et des acides nucléiques. / Cancer is considered as the leading cause of premature death in Canada. Taxanes (e.g. paclitaxel and docetaxel (DCTX)) are effective against a range of solid tumors including breast, lung, and ovarian malignancies. In addition, nucleic acids (e.g. antisense oligonucleotides (AON) and short interfering RNA (siRNA)) which are capable of selectively suppressing oncogenes involved in carcinogenesis are currently being investigated for the treatment of a wide variety of cancers. Although the activity of taxanes and nucleic acid drugs is well-established in human and/or animal models, several physicochemical and clinical issues still need to be addressed. Low aqueous solubility (i.e. taxanes), rapid degradation in the blood (i.e. nucleic acids), fast clearance, non-selectivity and toxicity to normal tissues are limiting factors to their effectiveness. Hence, many efforts have been focused on developing targeted polymeric delivery systems to overcome the problems associated with the current therapies. In this thesis, two types of polymeric micelles have been developed for the delivery of DCTX and nucleic acids. On the one hand, poly(ethylene oxide)-block-poly(butylene oxide/styrene oxide) micelles were tested for the first time to solubilize and protect DCTX from hydrolytic degradation. The polymers showed less toxicity than the surfactant used commercially to dissolve DCTX (i.e. polysorbate 80) and released the drug in a sustained fashion. On the other hand, two different systems of polyion complex micelles (PICM) were developed for the sustained release and intracellular delivery of nucleic acids. Novel poly(ethylene glycol) (PEG)-oligonucleotide conjugates were assessed to protect AON against degradation and release them in a sustained manner. When these conjugates were mixed with poly(amidoamine) (PAMAM) dendrimers, monodisperse PICM were formed. These PICM further slowed down AON release and significantly protected it against enzymatic degradation. In addition, the incorporation of poly(ethylene oxide)-block-poly(propyl methacrylate-co-methacrylic acid) was exploited to impart pH-sensitivity to PAMAM-based PICM. This system was composed of the previous copolymer mixed with PAMAM dendrimer. Such PICM were loaded with AON or siRNA targeting the Bcl-2 oncogene. Micelles uptake by the cancer cells was mediated by a monoclonal antibody fragment (i.e. Fab') positioned at the extremity of the PEG corona. Upon cellular uptake and protonation of the methacrylic acid units in the acidic endosomal environment, the micelles lost their corona, thereby exposing their positively-charged endosomolytic PAMAM/nucleic acid core. The targeted, pH-sensitive PICM were found to increase the intracellular bioavailability of the entrapped nucleic acids and knock down the Bcl-2 oncoprotein more than either non-targeted micelles or commercial PAMAM dendrimers. The polymeric nanocarriers reported in this thesis appear to be promising vehicles for the delivery of anticancer drugs and nucleic acids.