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5 Abstract Myeloid-derived suppressor cells (MDSC) are a very heterogeneous population of immature, activated myeloid progenitors of neutrophils, monocytes/macrophages and dendritic cells that have not differentiated into mature forms. A common feature of these cells is the ability to suppress immune responses of T cells, NK cells, and dendritic cells. It is known that MDSC accumulate under various pathological conditions, such as chronic inflammation or cancer. In breast cancer patients, the highest MDSC counts correlate with the occurrence of metastatic foci in lung tissue. The suppressive effects of MDSCs are associated with resistance to chemotherapy, reduced effectiveness of immunotherapy and overall poor prognosis of the disease. Therefore, many studies focus on MDSC. One possibility is the differentiation of MDSC into mature populations that lose their suppressive phenotype. In this work, we focused on modulation of MDSC activity by all-trans retinoic acid (ATRA), bound to a polymer conjugate based on N-(2-hydroxypropyl)methacrylamide (HPMA). ATRA is used in clinical practice for the treatment of acute promyelocytic leukemia, where the mechanism of action is the differentiation of pathological cells into more mature forms and thus the cessation of their proliferation. The binding of ATRA to the...

Mebendazole and other benzimidazole antihelmintics, such as albendazole, fenbendazole, or flubendazole, have been shown to possess antitumour activity, primarily due to their microtubule-disrupting activity. However, the extremely poor water-solubility of mebendazole and other benzimidazoles, resulting in very low bioavailability, is a serious drawback of this class of drugs. Thus, the investigation of their antitumour potential has been limited so far to administering repeated high doses given peroral (p.o.) or to using formulations, such as liposomes. Herein, we report a fully biocompatible, water-soluble, HPMA copolymer-based conjugate bearing mebendazole (P-MBZ; Mw 28–33 kDa) covalently attached through a biodegradable bond, enabling systemic administration. Such an approach not only dramatically improves mebendazole solubility but also significantly prolongs the half-life and ensures tumour accumulation via an enhanced permeation and retention (EPR) effect in vivo. This P-MBZ has remarkable cytostatic and cytotoxic activities in EL-4 T-cell lymphoma, LL2 lung carcinoma, and CT-26 colon carcinoma mouse cell lines in vitro, with corresponding IC50 values of 1.07, 1.51, and 0.814 µM, respectively. P-MBZ also demonstrated considerable antitumour activity in EL-4 tumour-bearing mice when administered intraperitoneal (i.p.), either as a single dose or using 3 intermittent doses. The combination of P-MBZ with immunotherapy based on complexes of IL-2 and anti-IL-2 mAb S4B6, potently stimulating activated and memory CD8+ T cells, as well as NK cells, further improved the therapeutic effect.

We explore a more concise process for the preparation of high-purity alumina and to address the problem of some conventional micro- and nano-adsorbents having difficulty in exposing their adsorption sites to target pollutants in solution due to the heavy aggregation of the adsorbent, which confers poor adsorption properties. The methods of using gamma-phase high-purity mesoporous alumina (HPMA), with its excellent adsorption properties and high adsorption rates of Congo Red, and of using lower-cost industrial aluminum hydroxide by direct aging and ammonium salt substitution were successfully employed. The results showed that the purity of HPMA was as high as 99.9661% and the total removal rate of impurities was 98.87%, a consequence of achieving a large specific surface area of 312.43 m2 g−1, a pore volume of 0.55 cm3 g−1, and an average pore diameter of 3.8 nm. The adsorption process was carried out at 25 °C, the concentration of Congo Red (CR) dye was fixed at 250 mg L−1 and the amount of adsorbent used was 100 mg. The HPMA sample exhibited an extremely fast adsorption rate in the first 10 min, with adsorption amounts up to 476.34 mg g−1 and adsorption efficiencies of 96.27%. The adsorption equilibrium was reached in about 60 min, at which time the adsorbed amount was 492.19 mg g−1 and the dye removal rate was as high as 98.44%. One-hundred milligrams of adsorbent were weighed and dispersed in 200-mL CR solutions with mass concentrations ranging from 50–1750 mg L−1 to study the adsorption isotherms. This revealed that the saturation adsorption capacity of the produced HPMA was 1984.64 mg g−1. Furthermore, the process of adsorbing Congo Red in the synthesized product was consistent with a pseudo-second order model and the Langmiur model. It is expected that this method of producing HPMA will provide a productive, easy and efficient means of treating toxic dyes in industrial wastewater.

Recently, the antitumor potential of benzimidazole anthelmintics, such as mebendazole and its analogues, have been reported to have minimal side effects, in addition to their well-known anti-parasitic abilities. However, their administration is strongly limited owing to their extremely poor solubility, which highly depletes their overall bioavailability. This study describes the design, synthesis, and physico-chemical properties of polymer-mebendazole nanomedicines for drug repurposing in cancer therapy. The conjugation of mebendazole to water-soluble and biocompatible polymer carrier was carried out via biodegradable bond, relying on the hydrolytic action of lysosomal hydrolases for mebendazole release inside the tumor cells. Five low-molecular-weight mebendazole derivatives, differing in their inner structure, and two polymer conjugates differing in their linker structure, were synthesized. The overall synthetic strategy was designed to enable the modification and polymer conjugation of most benzimidazole-based anthelmintics, such as albendazole, fenbendazole or albendazole, besides the mebendazole. Furthermore, the described methodology may be suitable for conjugation of other biologically active compounds with a heterocyclic N-H group in their molecules.

Polymer-based nanomedicines are extensively investigated in the field of cancer therapy to improve the poor aqueous solubility, limited therapeutic efficacy and off-target side-effects of chemotherapeutic agents. Particularly polymeric micelles, self-assembled nanostructures consisting of a core and a shell with a diameter between 10-100 nm have attracted a lot of attention. Generally, the hydrophobic core of the micelles can accommodate hydrophobic drugs, while the hydrophilic shell renders colloidal stability and protection against the immune system. Sufficient stability and drug retention of the nanoformulations in the blood circulation are prerequisites for achieving a favorable anticancer outcome. As previously demonstrated by our colleagues, polymeric micelles based on poly(ethylene glycol)-b-poly(N-2-benzoyloxypropyl methacrylamide) (mPEG-b-p(HPMA-Bz)), stabilized by π–π stacking interaction between the aromatic groups, are associated with excellent particle stability and good drug retention, thereby significantly improved the circulation kinetics and therapeutic efficacy of the loaded drug compared to the unformulated drug. In this project, nanoformulations based on mPEG-b-p(HPMA-Bz) polymer were further developed and optimized to obtain a manufacturing process that is potentially suitable for upscale production and subsequent clinical translation. Micelles were prepared by nanoprecipitation technique. The formulation and processing parameters influencing the nanostructures in both batch mode and microfluidics system were studied to tune the sizes in the range suitable for drug delivery (25-100 nm). The block copolymer properties (e.g, molecular weight, their hydrophilic-to-hydrophobic ratio, homopolymer content), organic solvents, aqueous phases, polymer concentrations and addition rates were varied. Interestingly, the continuous flow process demonstrated excellent control over the size and morphology with the possibility of large continuous production. Curcumin, a hydrophobic compound with a potential anticancer activity, was selected to encapsulate in these micelles. Stability studies in plasma showed no change in micelle size during 24 h incubation at 37 °C. While curcumin was released over time and the release rate was inversely dependent on the size of the hydrophobic block. In vivo studies in mice using the most stable formulation showed that the circulation time of curcumin was significantly shorter than that of the micelles. In contrast, the circulation time was around 5 times longer than has been reported for free curcumin. Therefore, mPEG-b-p(HPMA-Bz) micelles only marginally improved the circulation kinetics of the loaded curcumin compared to the free form and essentially performed as a solubilizer. Despite the solubilizing effect of these micelles, therapeutic efficacy was not observed in the experimental mice model, which might be related to the relatively low sensitivity of the tumor cells for curcumin. Further research is still required to show the potential of these curcumin-loaded micelles in cancer treatment.

The careful design of nanoparticles, in terms of size and morphology, is of great importance to developing effective drug delivery systems. The ability to precisely tailor nanoparticles in size and morphology during polymer self-assembly was therefore investigated. Four poly(ethylene glycol)-b-poly(N-2-benzoyloxypropyl methacrylamide) mPEG-b-p(HPMA-Bz) block copolymers with a fixed hydrophilic block of mPEG 5 kDa and a varying molecular weight of the hydrophobic p(HPMA-Bz) block (A: 17.1, B: 10.0, C: 5.2 and D: 2.7 kDa) were self-assembled into nanoparticles by nanoprecipitation under well-defined flow conditions, using microfluidics, at different concentrations. The nanoparticles from polymer A, increased in size from 55 to 90 nm using lower polymer concentrations and slower flow rates and even polymer vesicles were formed along with micelles. Similarly, nanoparticles from polymer D increased in size from 35 to 70 nm at slower flow rates and also formed vesicles along with micelles, regardless of the used concentration. Differently, polymers B and C mainly self-assembled into micelles at the different applied flow rates with negligible size difference. In conclusion, this study demonstrates that the self-assembly of mPEG-b-p(HPMA-Bz) block copolymers can be easily tailored in size and morphology using microfluidics and is therefore an attractive option for further scaled-up production activities.

Nitroksidni radikali su organske molekule koje imaju jedinstvena antioksidacijska svojstva te mogu suzbiti rast tumora. U in vivo uvjetima, ovi radikali ne mogu se učinkovito koristiti jer se nespecifično akumuliraju u zdravom tkivu te se brzo izlučuju iz organizma. U ovom radu istraživane su nanočestice temeljene na poli[N-(2-hidroksipropil)metakrilamidu] (poli(HPMA)) i kolesterolu koje sadrže TEMPONE radikal vezan hidrazonskom (RNP-H) i peptidnom vezom (RNP-P). Veličina i oblik nanočestica određeni su metodom krio-transmisijske elektronske mikroskopije. Utvrđeno je da su dobivene nanočestice sferičnog oblika prosječne veličine 25−50 nm. Otpuštanje radikala s nanočestica praćeno je metodom elektronske spinske rezonancije (ESR) u rasponu pH vrijednosti od 7,4 do 5,0. Pokazano je da u kiseloj sredini koja je karakteristična za tumorske stanice dolazi do brzog otpuštanja radikala vezanih hidrazonskom vezom dok nanočestice koje sadrže radikale vezane peptidnom vezom ostaju stabilne. Nasuprot tome, u fiziološkim uvjetima ne dolazi do značajnog oslobađanja radikala ni sa jedne vrste nanočestica. Ovim istraživanjem pokazano je da se polimerne nanočestice mogu koristiti kao nosači nitroksidnih radikala u novom pristupu terapiji tumora kada se žele izbjeći neželjene nuspojave slobodnih radikala i produljiti njihovo zadržavanje u organizmu., Nitroxide radicals are organic molecules that possess unique antioxidant properties and can suppress tumor growth. Under in vivo conditions, these radicals can not be used effectively because they accumulate nonspecifically in healthy tissue and are rapidly removed from the body. In this work, we investigated nanoparticles based on poly[N-(2-hydroxypropyl)methacrylamide] (poly(HPMA)) and cholesterol containing a TEMPONE radical bound by a hydrazone (RNP-H) and peptide bond (RNP-P) respectively. The size and shape of the nanoparticles were determined by cryo-transmission electron microscopy. Spherical nanoparticles with an average size of 25–50 nm were obtained. The release of radicals from the nanoparticles was investigated by electron spin resonance (ESR) spectroscopy in the pH range from 7.4 to 5.0. It was shown that the radicals bound by hydrazone bond are rapidly released in an acidic environment which is characteristic for tumor cells, while the nanoparticles containing radicals bound by peptide bond remain stable. In contrast, there is no significant release of radicals from both types of nanoparticles under physiological conditions. This research has shown that polymer nanoparticles can be used as a carriers of nitroxide radicals in a new approach to tumor therapy when we want to avoid unwanted side effects of free radicals and prolong their retention in the body.

Polymer-drug conjugates have several advantages in controlled drug delivery to inflammation as they can accumulate and release the drug in inflamed tissues or cells, which could circumvent the shortcomings of current therapy. To improve the therapeutic potential of polymer-drug conjugates in joint inflammation, we synthesized polymer conjugates based on N-(2-hydroxypropyl) methacrylamide) copolymers labeled with a near-infrared fluorescent dye and covalently linked to the anti-inflammatory drug dexamethasone (DEX). The drug was bound to the polymer via a spacer enabling pH-sensitive drug release in conditions mimicking the environment inside inflammation-related cells. An in vivo murine model of adjuvant-induced arthritis was used to confirm the accumulation of polymer conjugates in arthritic joints, which occurred rapidly after conjugate application and remained until the end of the experiment. Several tested dosage schemes of polymer DEX-OPB conjugate showed superior anti-inflammatory efficacy. The highest therapeutic effect was obtained by repeated i.p. application of polymer conjugate (3 &times, 1 mg/kg of DEX eq.), which led to a reduction in the severity of inflammation in the ankle by more than 90%, compared to 40% in mice treated with free DEX.

The careful design of nanoparticles, in terms of size and morphology, is of great importance to developing effective drug delivery systems. The ability to precisely tailor nanoparticles in size and morphology during polymer self-assembly was therefore investigated. Four poly(ethylene glycol)-b-poly(N-2-benzoyloxypropyl methacrylamide) mPEG-b-p(HPMA-Bz) block copolymers with a fixed hydrophilic block of mPEG 5 kDa and a varying molecular weight of the hydrophobic p(HPMA-Bz) block (A: 17.1, B: 10.0, C: 5.2 and D: 2.7 kDa) were self-assembled into nanoparticles by nanoprecipitation under well-defined flow conditions, using microfluidics, at different concentrations. The nanoparticles from polymer A, increased in size from 55 to 90 nm using lower polymer concentrations and slower flow rates and even polymer vesicles were formed along with micelles. Similarly, nanoparticles from polymer D increased in size from 35 to 70 nm at slower flow rates and also formed vesicles along with micelles, regardless of the used concentration. Differently, polymers B and C mainly self-assembled into micelles at the different applied flow rates with negligible size difference. In conclusion, this study demonstrates that the self-assembly of mPEG-b-p(HPMA-Bz) block copolymers can be easily tailored in size and morphology using microfluidics and is therefore an attractive option for further scaled-up production activities.Keywords: block copolymers; nanoparticles; micelles; polymersomes; HPMA; size control; nanoprecipitation; microfluidics; micromixer

Nitroksidni radikali su organske molekule koje imaju jedinstvena antioksidacijska svojstva te mogu suzbiti rast tumora. U in vivo uvjetima, ovi radikali ne mogu se učinkovito koristiti jer se nespecifično akumuliraju u zdravom tkivu te se brzo izlučuju iz organizma. U ovom radu istraživane su nanočestice temeljene na poli[N-(2-hidroksipropil)metakrilamidu] (poli(HPMA)) i kolesterolu koje sadrže TEMPONE radikal vezan hidrazonskom (RNP-H) i peptidnom vezom (RNP-P). Veličina i oblik nanočestica određeni su metodom krio-transmisijske elektronske mikroskopije. Utvrđeno je da su dobivene nanočestice sferičnog oblika prosječne veličine 25−50 nm. Otpuštanje radikala s nanočestica praćeno je metodom elektronske spinske rezonancije (ESR) u rasponu pH vrijednosti od 7, 4 do 5, 0. Pokazano je da u kiseloj sredini koja je karakteristična za tumorske stanice dolazi do brzog otpuštanja radikala vezanih hidrazonskom vezom dok nanočestice koje sadrže radikale vezane peptidnom vezom ostaju stabilne. Nasuprot tome, u fiziološkim uvjetima ne dolazi do značajnog oslobađanja radikala ni sa jedne vrste nanočestica. Ovim istraživanjem pokazano je da se polimerne nanočestice mogu koristiti kao nosači nitroksidnih radikala u novom pristupu terapiji tumora kada se žele izbjeći neželjene nuspojave slobodnih radikala i produljiti njihovo zadržavanje u organizmu.

This report describes the design, synthesis and evaluation of tumor-targeted polymer probes to visualize epidermal growth factor receptor (EGFR)-positive malignant tumors for successful resection via fluorescence guided endoscopic surgery. Fluorescent polymer probes of various molecular weights enabling passive accumulation in tumors via enhanced permeability and retention were prepared and evaluated, showing an optimal molecular weight of 200,000 g/mol for passive tumor targeting. Moreover, poly(N-(2-hydroxypropyl)methacrylamide)-based copolymers labeled with fluorescent dyes were targeted with the EGFR-binding oligopeptide GE-11 (YHWYGYTPQNVI), human EGF or anti-EGFR monoclonal antibody cetuximab were all able to actively target the surface of EGFR-positive tumor cells. Nanoprobes targeted with GE-11 and cetuximab showed the best targeting profile but differed in their tumor accumulation kinetics. Cetuximab increased tumor accumulation after 15 min, whereas GE 11 needed at least 4 h. Interestingly, after 4 h, there were no significant differences in tumor targeting, indicating the potential of oligopeptide targeting for fluorescence-navigated surgery. In conclusion, fluorescent polymer probes targeted by oligopeptide GE-11 or whole antibody are excellent tools for surgical navigation during oncological surgery of head and neck squamous cell carcinoma, due to their relatively simple design, synthesis and cost, as well as optimal pharmacokinetics and accumulation in tumors.

This study provides a synthesis of current scientific evidence on the ecological and socio-economic effects of highly protected marine areas (HPMAs), primarily in temperate waters. The aim was to establish if HPMAs can provide benefits beyond those afforded by other types of marine protected area (MPA). We identify critical interactions within and between ecological and socio-economic effects to help marine planners and managers to make informed decisions about the trade-offs of alternate management actions or measures for MPAs. Well-designed and enforced MPAs with high levels of protection (HPMAs) often provide conservation benefits within their boundaries beyond those afforded by other types of MPA. Much remains to be learned about the socio-economic effects of HPMAs. Empirical evidence to date suggests that potential benefits cannot all be maximised simultaneously because potentially conflicting trade-offs exist not only between but also within ecological and socio-economic effects. Marine planners and managers must be able to evaluate the impact and distribution of trade-offs for differing management regimes to make informed decisions about levels of protection required in MPAs to ensure sustainable use of marine resources and meet conservation objectives. One of the main challenges remains providing evidence of the societal benefits from restricting use in these areas.

Adenovirus-based vectors are playing an important role as efficacious genetic vaccines to fight the current COVID-19 pandemic. Furthermore, they have an enormous potential as oncolytic vectors for virotherapy and as vectors for classic gene therapy. However, numerous vector–host interactions on a cellular and noncellular level, including specific components of the immune system, must be modulated in order to generate safe and efficacious vectors for virotherapy or classic gene therapy. Importantly, the current widespread use of Ad vectors as vaccines against COVID-19 will induce antivector immunity in many humans. This requires the development of strategies and techniques to enable Ad-based vectors to evade pre-existing immunity. In this review article, we discuss the current status of genetic and chemical capsid modifications as means to modulate the vector–host interactions of Ad-based vectors.

Adenovirus-based gene transfer vectors are the most frequently used vector type in gene therapy clinical trials to date, and they play an important role as genetic vaccine candidates during the ongoing SARS-CoV-2 pandemic. Immediately upon delivery, adenovirus-based vectors exhibit multiple complex vector-host interactions and induce innate and adaptive immune responses. This can severely limit their safety and efficacy, particularly after delivery through the blood stream. In this review article we summarize two strategies to modulate Ad vector-induced immune responses: extensive genomic and chemical capsid modifications. Both strategies have shown beneficial effects in a number of preclinical studies while potential synergistic effects warrant further investigations.

Millions of people worldwide die of cancer every year. In the last decade, im- munotherapy offered new treatment options achieving long-lasting remissions in a number of patients. Several new immunotherapy-based drugs have been ap- proved by Food and Drug Administration. However, majority of patients either do not respond or soon relapse. Combination of therapies as well as exploring new immune checkpoints seems promising. This thesis focuses on the new immunotherapeutic target CD73. CD73 is membrane ectonucleotidase, widely expressed on the regulatory leukocytes and on cancer cells. The enzymatically active CD73 contributes to the tumour mi- croenvironment by production of immunosuppressive adenosine. This novel im- mune checkpoint is being intensively studied. This thesis aims on development of new approaches for targeting and inhibition of CD73. Soluble recombinant CD73 (rhCD73) was prepared in mammalian expression system and transfectants stably expressing membrane-bound CD73 were prepared as well. Inhibitors necessary for both of my goals have been designed based on published inhibitor of CD73. Development and evaluation of novel antibody mimetic for CD73 characteri- sation was done. The so-called iBody, HPMA polymer conjugate decorated with CD73 inhibitor for targeting, fluorophore for...

U ovom radu ispitano je otpuštanje nitroksilnih radikala koji se koriste za liječenje bolesti uzrokovanih oksidacijskim stresom sa polimernog nosača temeljenog na poli[N-(2-hidroksipropil)metakrilamidu] (poly(HPMA)). 4-okso-2,2,6,6-tetrametilpiperidin-1-oksil (TEMPONE) radikali vezani su na poly(HPMA) nosač preko razmaknice koja sadrži pH-osjetljivu hidrazonsku vezu. Priređeni hidrofilni polimer otopljen je u fosfatnom puferu pri različitim pH vrijednostima (7,4, 6,8, 6,0, 5,0). Hidrodinamički radijus molekula nosača u otopinama određen je metodom dinamičkog raspršenja svjetlosti (DLS). Pokazano je da su molekule poly(HPMA) u otopinama poprimile konformaciju statističkog klupka hidrodinamičkog radijusa 3,5 nm sa širokom razdiobom veličina. Oslobađanje nitroksilnih radikala sa poly(HPMA) nosača određivano je metodom elektronske spinske rezonancije (ESR). Utvrđeno je da se radikali brzo oslobađaju sa nosača pri uvjetima karakterističnim za endosome tumorskih stanica (pH = 5,0) dok pri fiziološkim uvjetima (pH = 7,4) radikali uglavnom ostaju vezani na nosaču. Na ovaj način postiže se kontrolirano otpuštanje radikala u tumoru te se izbjegavaju neželjeni učinci nitroksilnih radikala na ostale organe i tkiva., In this thesis, the release of nitroxyl radicals used to treat diseases caused by oxidative stress from poly[N-(2-hydroxypropyl)methacrylamide] (poly(HPMA)) is investigated. 4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPONE) radicals are attached to a poly(HPMA) carrier by a spacer containing pH-sensitive hydrazone linkage. The prepared hydrophilic polymer is dissolved in phosphate buffer at various pH values (7.4, 6.8, 6.0, 5.0). The hydrodynamic radius of the carrier molecules in solutions was determined by the dynamic light scattering method (DLS). It has been shown that the poly(HPMA) molecules in the solutions obtained random coil conformation with the hydrodynamic radius of 3.5 nm and broad size distribution. The release of nitroxyl radicals from poly(HPMA) carrier was determined by the electron spin resonance (ESR) method. It was found that the radicals are quickly released from the carrier under conditions characteristic for the endosomal tumor cells (pH = 5.0) while at physiological conditions (pH = 7.4) the radicals remained largely bound to the carrier. In this way, controlled release of the radicals in the tumor can be achieved and the unwanted effects of nitroxyl radicals on other organs and tissues are avoided.

Antibodies are indispensable tools for biomedicine and anticancer therapy. Nevertheless, their use is compromised by high production costs, limited stability, and difficulty of chemical modification. The design and preparation of synthetic polymer conjugates capable of replacing antibodies in biomedical applications such as ELISA, flow cytometry, immunocytochemistry, and immunoprecipitation is reported. The conjugates, named “iBodies”, consist of an HPMA copolymer decorated with low‐molecular‐weight compounds that function as targeting ligands, affinity anchors, and imaging probes. We prepared specific conjugates targeting several proteins with known ligands and used these iBodies for enzyme inhibition, protein isolation, immobilization, quantification, and live‐cell imaging. Our data indicate that this highly modular and versatile polymer system can be used to produce inexpensive and stable antibody substitutes directed toward virtually any protein of interest with a known ligand.

In this work, activated ester chemistry is employed to synthesize biocompatible and readily functionalizable polymersomes. Via aminolysis of pentafluorophenyl methacrylate-based precursor polymers, an N-(2-hydroxypropyl) methacrylamide (HPMA)-analog hydrophilic block is obtained. The precursor polymers can be versatile functionalized by simple addition of suitable primary amines during aminolysis as demonstrated using a fluorescent dye. Vesicle formation is proven by cryoTEM and light scattering. High encapsulation efficiencies for hydrophilic cargo like siRNA are achieved using dual centrifugation and safe encapsulation is demonstrated by gel electrophoresis. In vitro studies reveal low cytotoxicity and no protein adsorption-induced aggregation in human blood serum occurs, making the vesicles interesting candidates as nanosized drug carriers.

Statistical copolymers of N-(2-hydroxypropyl) methacrylamide (HPMA) and the dendronic methacrylic monomer 2-(3-(Bis(2-(diethylamino)ethyl)amino)propanamido)ethyl methacrylate (TEDETAMA, derived from N,N,N′,N′-tetraethyldiethylenetriamine, TEDETA), were synthesized through radical copolymerization and evaluated in vitro as non-viral gene carriers. Three copolymers with nominal molar percentages of HPMA of 25%, 50% and 75% were prepared and studied comparatively to the positive controls poly-TEDETAMA and hyperbranched polyethyleneimine (PEI, 25 kDa). Their ability to complex DNA at different N/P molar ratios, from 1/1 up to 8/1, was determined through agarose gel electrophoresis and Dynamic Light Scattering. The resulting complexes (polyplexes) were characterized and evaluated in vitro as possible non-viral gene carriers for Swiss-3T3 fibroblasts, using luciferase as reporter gene and a calcein cytocompatibility assay. All the copolymers, except the one with highest HPMA proportion (75 molar %) at the lowest N/P ratio, condensed DNA to a particle size between 100 and 300 nm. The copolymers with 25 and 50 molar % of HPMA displayed higher transfection efficiency and cytocompatibility than the positive controls poly-TEDETAMA and PEI. A higher proportion of HPMA (75 molar %) led to copolymers that displayed very low transfection efficiency, despite their full cytocompatibility even at the highest N/P ratio. These results indicate that the statistical combination of TEDETAMA and HPMA and its fine compositional tuning in the copolymers may fulfill the fine balance of transfection efficiency and cytocompatibility in a superior way to the control poly-TEDETAMA and PEI.

The work presented here represents three separate research projects. Each explores different approaches to the same problem: how can artificial macromolecules be used to target specific intracellular sites in living organisms, and how may that technology be exploited to treat different disease states? Here, the copolymers based on pHPMA [poly N-(2-hydroxypropyl)methacrylamide)] were used to demonstrate cellular and subcellular targeting in a cancer model. Used as carriers for low molecular weight drugs, pHPMA copolymers may possess great potential for increasing the efficacy of drugs, while also decreasing side effects resulting from drug exposure to healthy tissues and cells. In the first project, antibody Fab’ fragments were dimerized using a multifunctionalized PEG (poly(ethylene glycol)) crosslinker to create a targeting module for pHPMA/drug carriers. The linker was semitrifunctional, able to crosslink 2 Fab’ antibody fragments, which could then be attached to an HPMA-doxorubicin anticancer drug carrier. Monoclonal antibody Fab’ fragments were chosen that target ovarian carcinoma cells. Using this strategy, the conjugates are selectively uptaken by cancer cells, whereby free doxorubicin is released. In the second project, HPMA copolymers were created with a terminal mitochondriotropic chemical moiety, TPP (triphenylphosphonium). Lipophilic cations are used to traffic low molecular weight drugs to mitochondria. Studies had reported that terminal TPP can also traffic high molecular weight, uncharged, linear macromolecules into mitochondria, and enable delivery to the cytosol via direct transduction through the plasma membrane. Semitelechelic TPP-HPMA copolymers were synthesized to determine if this effect could be applied to HPMA copolymer drug conjugates. In the third project, the flexibility of HPMA polymerization chemistry was exploited to create a large “library” or array of different HPMA copolymers with a wide range of chemical properties. Copolymers were made incorporating a variety of cationic, anionic, and hydrophobic sidechains. This array of copolymers was incubated and microinjected directly into the cytoplasm of living cells. The resulting uptake and/or intracellular distribution was observed using time-lapse confocal microscopy and flow cytometry. Using HPMA copolymers as a model, this work functioned as a general survey for cellular uptake, and the intracellular distribution/trafficking that can be expected when artificial macromolecules are internalized into living cells.

In dieser Arbeit wurden makromolekulare Wirkstoffträger basierend auf N-(2-Hydroxypropyl)methacrylamid (pHPMA) hinsichtlich ihrer Aufnahme in die Tumorzelllinien AT-1 und Walker-256 untersucht. Es wurden dafür je eine kurze und längere Polymerkette von strukturell verschiedenen pHPMA-Varianten (Homopolymere, Random-Copolymere, Block-Copolymere) näher betrachtet. In Vorversuchen wurde gezeigt, dass diese sich hinsichtlich der Biodistribution und Tumorakkumulation in beiden Tumormodellen der Ratte unterschieden. Darum wurden zunächst diese beiden Tumormodelle in vivo näher hinsichtlich der Gefäßdichte, des pO2, des extrazellulären pH-Wertes und weiterer Parameter charakterisiert. In Zellkulturversuchen wurde dann der Einfluss eines hypoxischen und azidotischen Milieus auf die Aufnahme der Polymere in beide Zelllinien untersucht. Zudem wurden mögliche Endozytosewege der Polymere durch Inhibitorstudien und der Einfluss der Hypoxie und Azidose auf diese Endozytosewege näher betrachtet., In this study, macromolecular drug carriers based on N-(2-hydroxypropyl)methacrylamide (pHPMA) were investigated for their uptake into the tumor cell lines AT-1 and Walker-256. A short and a longer polymer chain of structurally different pHPMA variants (homopolymers, random copolymers, block copolymers) were examined in detail. In preliminary experiments, it was shown that these differed in terms of biodistribution and tumor accumulation in both rat tumor models. Therefore, these two tumor models were characterized in vivo with respect to vessel density, pO2, extracellular pH and other parameters. In cell culture experiments, the impact of a hypoxic and acidotic environment on the uptake of polymers in both cell lines was determined. In addition, possible endocytosis pathways of the polymers were investigated by inhibitor studies and the impact of hypoxia and acidosis on these endocytosis pathways.

Polymeric micelles based on HPMA [ N -(2-hydroxypropyl) methacrylamide] polymers were recently evaluated as drug delivery systems of several anticancer drugs. The development of polymeric micelles to solubilize R-(+)-MRJF4, a potential anticancer prodrug, is reported in this paper. Two different amphiphilic block copolymers based on PEG-HPMA [( ω -methoxypoly (ethylene glycol)- b -( N -(2-benzoyloxy-propyl) methacrylamide)- co -( N -(2-lactoyloxypropyl) methacrylamide) (PEG-HPMA-Bz-L) and ( ω -methoxy poly (ethylene glycol)- b -( N -(2-benzoyloxypropyl) methacrylamide (PEG-HPMA-Bz)] were synthesized and investigated for this purpose. Results showed that both polymers were able to efficiently solubilize the drug at concentrations of 2 and 4 mg/mL and polymer concentration of 9 mg/mL yielding polymeric micelles with a size of 53–83 nm. Release studies showed that the formulation obtained using PEG-HPMA-Bz-L slowly released R-(+)-MRJF4 for 7–8 days. Moreover, cytotoxicity studies performed on C6 glioma cells revealed that, after 48 h, R-(+)-MRJF4-loaded PEG-HPMA-Bz and PEG-HPMA-Bz-L micelles possessed a higher antiproliferative activity when compared to free R-(+)-MRJF4, implying that the formulations could be internalized by the cells. Taken together, our results suggest that PEG-HPMA-Bz-L polymeric micelles are interesting to optimize the therapeutic efficacy of R-(+)-MRJF4.

Polymeric micelles have been under extensive investigation during the past years as drug delivery systems, particularly for anticancer drugs. They are formed by the self-assembly of amphiphilic block copolymers in aqueous solutions and have a spherical shape and a size in the nano-range (

Polymer-based drug delivery systems represent one of the promising strategies for successful tumor treatment. Conjugation of a low-molecular-weight drug to a syn- thetic polymer carrier enables targeted drug delivery to tumor tissue/cells and limited systemic toxicity of the drug. The conjugates show extended circulation time, and preferentially accumulate in tumor tissue due to the Enhanced Permeability and Re- tention (EPR) effect. The EPR effect depends on a structural anomaly in tumor neovasculature, and vasodilators were shown to enhance the EPR effect via an in- crease of blood supply in the tumor. Polymer drug carriers based on water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) benefit from variable architecture, drug loading and controlled release. HPMA-based conjugates with cancerostatics have al- ready proved high anti-tumor activity, inducing complete tumor regression followed by resistance to a second tumor challenge in experimental murine models. Three HPMA-based conjugates with organic nitrates (labeled 1, 2, and 3) were pre- pared as polymer donors of nitric oxide (NO) with the aim to intensify the EPR effect, thereby enhancing accumulation of co-administered macromolecular cancerostatics in the tumor. In this study, the conjugates were non-toxic to cancer cells and did not potentiate...

The direct intratumoral (i.t.) injection of anticancer agents has been evaluated extensively in the past few decades. Thus far, however, it has failed to become established as an alternative route of administration in routine clinical practice. In the present report, the impact of i.t. injection on the biodistribution and the therapeutic potential of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based drug delivery systems was investigated. It was found that, compared to intravenous injection, both the tumor concentrations and the tumor-to-organ ratios of carriers improved substantially. In addition, compared to intravenously and intratumorally applied free doxorubicin and to intravenously applied poly(HPMA)-glycylphenylalanylleucylglycine-doxorubicin, intratumorally injected poly(HPMA)-glycylphenylalanylleucylglycine-doxorubicin presented a significantly increased antitumor efficacy, as well as an improved therapeutic index. Based on these findings, we propose intratumorally injected carrier-based chemotherapy as an interesting alternative to routinely used chemotherapy regimens and routes of administration.

In this work, the cationic monomer N-ethyl pyrrolidine methacrylamide (EPA) was copolymerized with the neutral monomer N-hydroxypropyl methacrylamide (HPMA) at different molar ratios obtaining linear random copolymers that were characterized and evaluated in vitro as non-viral gene carriers using murine Swiss 3T3 fibroblasts. The copolymers with excess or equimolar amount of EPA were able to complex DNA forming stable polyplexes with an average size between 50 and 200 nm, while the copolymers with an excess of HPMA do not. Cell viability was shown to depend on the EPA/HPMA molar ratio, exhibiting the equimolar copolymer poly (EPA-co-HPMA) 50:50 (EPA50) a full cytocompatibility, similar to the HPMA-rich systems. This copolymer EPA50 has also shown significantly higher transfection levels than the systems with other compositions and the positive controls poly l-lysine (PLL) and poly EPA (pEPA). This statistical equimolar copolymer EPA50 has unique properties related to its composition and microstructure, which allows it to complex DNA, showing an excellent biocompatibility and high transfection efficiency.

The most adopted methods to produce polymer nanoparticles (NPs) for medical and pharmaceutical applications use surfactants that are toxic and physically adsorbed to the NPs, with the risk of desorption and insurgence of side effects. A valid alternative is represented by the use of surfmers, reactive surfactants that are chemically linked to the polymer chains, thus avoiding the release of toxic species. In this case, the lack of biodegradable surfmers introduces the concern of polymer accumulation into the body. In this work, biodegradable, poly(ethylene glycol) (PEG)ylated N-(2-hydroxypropyl) methacrylamide (HPMA) based surfmers are synthesized and used to stabilize lipophilic NPs. In particular, the NP core is made from a macromonomer comprising a poly(lactic acid) (PLA) chain functionalized with HPMA double bond. The NP forming polymer chains are then constituted by a uniform poly(HPMA) backbone that is biocompatible and water soluble and hydrolysable PEG and PLA pendants assuring the complete degradability of the polymer. The stability provided to these NPs by the synthesized surfmers is studied in the cases of both emulsion free radical polymerization and solution free radical polymerization followed by the flash nanoprecipitation of the obtained amphiphilic copolymers.

This diploma thesis brings new data about design, synthesis, physico-chemical characterisation and biological efficacy of the novel star-like HPMA-based conjugates intended for treatment of solid tumors. Recently, many different water-soluble drug delivery systems based on N-(2- hydroxypropyl)methacrylamide (HPMA) copolymers have been described. Here, we report synthesis and physico-chemical characterisation of high molecular weight star-like HPMA- based polymer carriers with low polydispersity prepared by controlled grafting of HPMA copolymers onto PAMAM dendrimer core. With the aim to keep the polydispersity of drug delivery system as low as possible, reversible Addition-Fragmentation Chain Transfer (RAFT) polymerisation was used for HPMA-based polymer precursor preparation. The end groups of the polymer presursors was afterwards used for grafting using carbodidimide condensation reaction or copper free click chemistry on polyamidoamine (PAMAM) dendrimers resulting in a formation of star-like high-molecular-weight (HMW) drug carriers. Described synthetic procedure provided preparation of star-like HMW drug carriers with Mw between 1.105 - 3.105 g/mol and narrow distribution of Mw. The model drug, doxorubicin (Dox), was attached to the hydrazide group containing polymer cariers by pH- sensitive...

vi ABSTRACT IL-2 has been used in cancer therapy and also for other applications like treatment of chronic viral infections or as an adjuvant for vaccines. However, treatment with IL-2 is rather difficult due to its severe side effects. These toxicities, associated with high-dose treatment necessary for IL-2 to function, have been found the most limiting factor for IL- 2 applications. Further, particular anti-IL-2 monoclonal antibodies (mAb) can actually increase biological activity of IL-2 rather than block it. Binding of IL-2 to anti-IL-2 mAb creates a superagonistic immunocomplexes which have dramatically higher and selective biological activity in comparison to free IL-2 in vivo. Such approach may finally over- come the difficulties associated with administration of IL-2, thus opening brand new scopes for IL-2 and its application not only in the field of tumor therapy. We have shown that IL-2 immunocomplexes composed of IL-2 and anti-IL-2 mAb S4B6 (IL-2/S4B6) stimulate predominantly cells expressing CD122 and CD132 (dimeric IL-2 receptor), i.e. NK and MP CD8+ T cells, with Treg,  T and NKT cells being expanded as well. IL-2/S4B6 are able to drive the expansion of activated naive CD8+ T cells into functional memory-like CD8+ T cells. Moreover, these immunocomplexes exert therapeu- tical potential alone...

The use of ?-sheets as building blocks for biomaterials is already firmly established. In particular, self-assembled ?-sheet peptides are promising for engineering new fibrous nanostructures and hydrogels. Peptide-synthetic polymer hybrids are especially attractive since they combine the advantages of biomolecular recognition and functional properties of peptides with the low cost and easy fabrication of polymers. Significant developments in the area have included ?-sheet fibrillar networks, and selfassembled hybrid hydrogels, which add further control and utility to these systems. The studies described in this dissertation dealt with the design and evaluation of novel nanofibrous and hydrogel materials based on poly(HPMA)-?-sheet copolymers and their application as scaffolds for bone tissue engineering. In the first part of this research, the effect of conjugating poly(HPMA) to a ?-sheet peptide via thiol-maleimide chemistry was estimated. The ability of the peptide to adopt a ?-sheet conformation could be imposed in the hybrid at basic pH, through electrostatic interactions between the oppositely charged amino acid residues in the sequence. Hierarchically organized structures, such as micrometer long fibrils, were obtained. In the second part, formation of fibril-like nanostructures was demonstrated for ?-sheet peptides conjugated as grafts to poly(HPMA).

The direct intratumoral (i.t.) injection of anticancer agents has been evaluated extensively in the past few decades. Thus far, however, it has failed to become established as an alternative route of administration in routine clinical practice. In the present report, the impact of i.t. injection on the biodistribution and the therapeutic potential of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based drug delivery systems was investigated. It was found that, compared to intravenous injection, both the tumor concentrations and the tumor-to-organ ratios of carriers improved substantially. In addition, compared to intravenously and intratumorally applied free doxorubicin and to intravenously applied poly(HPMA)-glycylphenylalanylleucylglycine-doxorubicin, intratumorally injected poly(HPMA)-glycylphenylalanylleucylglycine-doxorubicin presented a significantly increased antitumor efficacy, as well as an improved therapeutic index. Based on these findings, we propose intratumorally injected carrier-based chemotherapy as an interesting alternative to routinely used chemotherapy regimens and routes of administration.

Copolymers of camptothecin (CPT) and [N-(2-hydroxypropyl) methacrylamide] (HPMA) are novel anticancer drugs that show improved pharmacological profile in animal models as compared to the free drug CPT. We investigate here the aggregation properties of a HPNIA-glycyl-6-aminohexanoyl-glycyl-CPT copolymer (similar to20,000 Da). The molecular size of HPMA-copolymer CPT is followed over 5 orders of magnitudes of concentration in isotonic buffer by measuring either the time resolved fluorescence anisotropy (FA) of CPT or the autocorrelation function of the light scattered by the copolymer. A detailed analysis of these data suggests the presence of elongated structures with axial ratio similar to3 in the range 0.1-0.5 mug/ml and aggregates with association number higher than 2 in more concentrated solutions (up to 10 mg/ml). The binding affinity of HPMA-copolymer CPT for serum albumin is inversely dependent on the degree of aggregation of the copolymer. We also show that the copolymer concentration in plasma from mice treated with an active, non-toxic, dose of HPMA-copolymer CPT, decreases from 3 to 0.01 mg/ml in 72 h. In the same range of concentrations in vitro, we do not detect hydrophobic aggregates of polymers with high (>3) association number. Our study indicates that the circulating HPMA-copolymer CPT in mice should not undergo extensive aggregation and should interact with serum albumin more weakly than free CPT. (C) 2004 Elsevier B.V. All rights reserved.

The direct intratumoral (i.t.) injection of anticancer agents has been evaluated extensively in the past few decades. Thus far, however, it has failed to become established as an alternative route of administration in routine clinical practice. In the present report, the impact of i.t. injection on the biodistribution and the therapeutic potential of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based drug delivery systems was investigated. It was found that, compared to intravenous injection, both the tumor concentrations and the tumor-to-organ ratios of carriers improved substantially. In addition, compared to intravenously and intratumorally applied free doxorubicin and to intravenously applied poly(HPMA)-glycylphenylalanylleucylglycine-doxorubicin, intratumorally injected poly(HPMA)-glycylphenylalanylleucylglycine-doxorubicin presented a significantly increased antitumor efficacy, as well as an improved therapeutic index. Based on these findings, we propose intratumorally injected carrier-based chemotherapy as an interesting alternative to routinely used chemotherapy regimens and routes of administration.

Background We recently synthesized a hydrophilic polymer, poly(N-(2-hydroxypropyl)methacrylamide-co-N-(3-azidopropyl)methacrylamide), p(HPMA-co-AzMA), by RAFT polymerization using a novel azide-containing methacrylamide monomer that through a post modification strategy using click chemistry enabled facile preparation of a panel of versatile and well-defined bioconjugates. In this work we screen a panel of different molecular weight (Mw) fluorescently tagged p(HPMA-co-AzMA) in healthy mice, by live bioimaging, to select an extended circulatory half-life material for investigating joint accumulation in a murine collagen antibody-induced arthritis model. Findings Fluorescence image analysis revealed half-lifes of