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Nanomedicines are considered next generation therapeutics with advanced therapeutic properties and reduced side effects. Herein, we introduce tailored linear and star-like water-soluble nanosystems as stimuli-sensitive nanomedicines for the treatment of solid tumors or hematological malignancies. The polymer carrier and drug pharmacokinetics were independently evaluated to elucidate the relationship between the nanosystem structure and its distribution in the body. Positron emission tomography and optical imaging demonstrated enhanced tumor accumulation of the polymer carriers in 4T1-bearing mice with increased tumor-to-blood and tumor-to-muscle ratios. Additionally, there was a significant accumulation of doxorubicin bound to various polymer carriers in EL4 tumors, as well as excellent in vivo therapeutic activity in EL4 lymphoma and moderate efficacy in 4T1 breast carcinoma. The linear nanomedicine showed at least comparable pharmacologic properties to the star-like nanomedicines regarding doxorubicin transport. Therefore, if multiple parameters are considered such as its optimized structure and simple and reproducible synthesis, this polymer carrier system is the most promising for further preclinical and clinical investigations.

Recently, we have developed synthetic polymer-based antibody mimetics called iBodies that can successfully replace antibodies in many biochemical applications including ELISA (enzyme-linked immunosorbent assay), flow cytometry, immunocytochemistry, affinity purification, and confocal microscopy. In this study, we focused primarily on the effect of a linker between the polymer backbone and biotin, and then on the influence of the number of biotins per polymer chain on the efficacy of the ELISA or pull-down assays. In addition, the biotin position on the polymer chain was investigated. Moreover, we developed a novel chain transfer agent suitable for RAFT polymerization, which enables the functionalization of specifically the polymer precursors and simplifies the synthesis of semitelechelic antibody mimetic materials. By employing optimized iBodies the sensitivity of the ELISA with a lengthened linker between the polymer backbone and biotin was increased up to 5 times. Importantly, we found that one biotin at the end of the polymer chain can replace up to 12 biotins located along the polymer chain and maintain the signal level in the ELISA, as well as in the pull-down assay.

The derivative of protease inhibitor ritonavir (5-methyl-4-oxohexanoic acid ritonavir ester; RD) was recently recognized as a potent P-gp inhibitor and cancerostatic drug inhibiting the proteasome and STAT3 signaling. Therefore, we designed high-molecular-weight HPMA copolymer conjugates with a PAMAM dendrimer core bearing both doxorubicin (Dox) and RD (Star-RD + Dox) to increase the circulation half-life to maximize simultaneous delivery of Dox and RD into the tumor. Star-RD inhibited P-gp activity, potently sensitizing both low- and high-P-gp-expressing cancer cells to the cytostatic and proapoptotic activity of Dox in vitro. Star-RD + Dox possessed higher cytostatic and proapoptotic activities compared to Star-Dox and the equivalent mixture of Star-Dox and Star-RD in vitro. Star-RD + Dox efficiently inhibited STAT3 signaling and induced caspase-3 activation and DNA fragmentation in cancer cells in vivo. Importantly, Star-RD + Dox was found to have superior antitumor activity in terms of tumor growth inhibition and increased survival of mice bearing P-gp-expressing tumors.

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.

Aqueous Cu-RDRP of N-(2-hydroxypropyl) methacrylamide, a monomer with important biomedical applications, was optimized with respect to monomer conversion (82–99%), product dispersity (

Recently, numerous polymer materials have been employed as drug carrier systems in medicinal research, and their detailed properties have been thoroughly evaluated. Water-soluble polymer carriers play a significant role between these studied polymer systems as they are advantageously applied as carriers of low-molecular-weight drugs and compounds, e.g., cytostatic agents, anti-inflammatory drugs, antimicrobial molecules, or multidrug resistance inhibitors. Covalent attachment of carried molecules using a biodegradable spacer is strongly preferred, as such design ensures the controlled release of the drug in the place of a desired pharmacological effect in a reasonable time-dependent manner. Importantly, the synthetic polymer biomaterials based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers are recognized drug carriers with unique properties that nominate them among the most serious nanomedicines candidates for human clinical trials. This review focuses on advances in the development of HPMA copolymer-based nanomedicines within the passive and active targeting into the place of desired pharmacological effect, tumors, inflammation or bacterial infection sites. Specifically, this review highlights the safety issues of HPMA polymer-based drug carriers concerning the structure of nanomedicines. The main impact consists of the improvement of targeting ability, especially concerning the enhanced and permeability retention (EPR) effect.

Nanomedicines are a novel class of therapeutics that benefit from the nano dimensions of the drug carrier. These nanosystems are highly advantageous mainly within cancer treatment due to their enhanced tumor accumulation. Monolayer tumor cells frequently used in routine preclinical assessment of nanotherapeutics do not have a spatial structural architecture that allows the investigation of the penetration of nanomedicines to predict their behavior in real tumor tissue. Therefore, tumor spheroids from colon carcinoma C26 cells and glioblastoma U87-MG cells were used as 3D in vitro models to analyze the effect of the inner structure, hydrodynamic size, dispersity, and biodegradability of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based nanomedicines carrying anticancer drug pirarubicin (THP) on the penetration within spheroids. While almost identical penetration through spheroids of linear and star-like copolymers and also their conjugates with THP was observed, THP penetration after nanomedicines application was considerably deeper than for the free THP, thus proving the benefit of polymer carriers. The cytotoxicity of THP-polymer nanomedicines against tumor cell spheroids was almost identical as for the free THP, whereas the 2D cell cytotoxicity of these nanomedicines is usually lower. The nanomedicines thus proved the enhanced efficacy within the more realistic 3D tumor cell spheroid system.

Drug repurposing is a promising strategy for identifying new applications for approved drugs. Here, we describe a polymer biomaterial composed of the antiretroviral drug ritonavir derivative (5-methyl-4-oxohexanoic acid ritonavir ester; RD), covalently bound to HPMA copolymer carrier via a pH-sensitive hydrazone bond (P-RD). Apart from being more potent inhibitor of P-glycoprotein in comparison to ritonavir, we found RD to have considerable cytostatic activity in six mice (IC50 ~ 2.3–17.4 μM) and six human (IC50 ~ 4.3–8.7 μM) cancer cell lines, and that RD inhibits the migration and invasiveness of cancer cells in vitro. Importantly, RD inhibits STAT3 phosphorylation in CT26 cells in vitro and in vivo, and expression of the NF-κB p65 subunit, Bcl-2 and Mcl-1 in vitro. RD also dampens chymotrypsin-like and trypsin-like proteasome activity and induces ER stress as documented by induction of PERK phosphorylation and expression of ATF4 and CHOP. P-RD nanomedicine showed powerful antitumor activity in CT26 and B16F10 tumor-bearing mice, which, moreover, synergized with IL-2-based immunotherapy. P-RD proved very promising therapeutic activity also in human FaDu xenografts and negligible toxicity predetermining these nanomedicines as side-effect free nanosystem. The therapeutic potential could be highly increased using the fine-tuned combination with other drugs, i.e. doxorubicin, attached to the same polymer system. Finally, we summarize that described polymer nanomedicines fulfilled all the requirements as potential candidates for deep preclinical investigation.

In this review we summarize several synthetic approaches to the advanced synthesis of star-like polymer-based drug carriers. Moreover, their application as nanomedicines for therapy or the diagnosis of neoplastic diseases and their biodistribution are reviewed in detail. From a broad spectrum of star-like systems, we focus only on fully water-soluble systems, mainly based on poly(ethylene glycol) or N-(2-hydroxypropyl)methacrylamide polymer and copolymer arms and polyamidoamine dendrimers serving as the core of the star-like systems.

Glutamate carboxypeptidase II (GCPII) is a membrane protease overexpressed by prostate cancer cells and detected in the neovasculature of most solid tumors. Targeting GCPII with inhibitor-bearing nanoparticles can enable recognition, imaging, and delivery of treatments to cancer cells. Compared to methods based on antibodies and other large biomolecules, inhibitor-mediated targeting benefits from the low molecular weight of the inhibitor molecules, which are typically stable, easy-to-handle, and able to bind the enzyme with very high affinity. Although GCPII is established as a molecular target, comparing previously reported results is difficult due to the different methodological approaches used. In this work, we investigate the robustness and limitations of GCPII targeting with a diverse range of inhibitor-bearing nanoparticles (various structures, sizes, bionanointerfaces, conjugation chemistry, and surface densities of attached inhibitors). Polymer-coated nanodiamonds, virus-like particles based on bacteriophage Qβ and mouse polyomavirus, and polymeric poly(HPMA) nanoparticles with inhibitors attached by different means were synthesized and characterized. We evaluated their ability to bind GCPII and interact with cancer cells using surface plasmon resonance, inhibition assay, flow cytometry, and confocal microscopy. Regardless of the diversity of the investigated nanosystems, they all strongly interact with GCPII (most with low picomolar K

Peptide display methods are a powerful tool for discovering new ligands of pharmacologically relevant targets. However, the selected ligands often suffer from low affinity. Using phage display, we identified a new bicyclic peptide binder of prostate-specific membrane antigen (PSMA), a metalloprotease frequently overexpressed in prostate cancer. We show that linking multiple copies of a selected low-affinity peptide to a biocompatible water-soluble N-(2-hydroxypropyl)methacrylamide copolymer carrier (iBody) improved binding of the conjugate by several orders of magnitude. Furthermore, using ELISA, enzyme kinetics, confocal microscopy, and other approaches, we demonstrate that the resulting iBody can distinguish between different conformations of the target protein. The possibility to develop stable, fully synthetic, conformation-selective antibody mimetics has potential applications for molecular recognition, diagnosis and treatment of many pathologies. This strategy could significantly contribute to more effective drug discovery and design.

Multidrug resistance (MDR) is often caused by the overexpression of efflux pumps, such as ABC transporters, in particular, P-glycoprotein (P-gp). Here, we investigate the di- and tri- block amphiphilic polymer systems based on polypropylene glycol (PPO) and copolymers of (N-(2-hydroxypropyl)methacrylamide) (PHPMA) as potential macromolecular inhibitors of P-gp, and concurrently, carriers of drugs, passively targeting solid tumors by the enhanced permeability and retention (EPR) effect. Interestingly, there were significant differences between the effects of di- and tri- block polymer-based micelles, with the former being significantly more thermodynamically stable and showing much higher P-gp inhibition ability. The presence of Boc-protected hydrazide groups or the Boc-deprotection method did not affect the physico-chemical or biological properties of the block copolymers. Moreover, diblock polymer micelles could be loaded with free PPO containing 5&ndash, 40 wt % of free PPO, which showed increased P-gp inhibition in comparison to the unloaded micelles. Loaded polymer micelles containing more than 20 wt % free PPO showed a significant increase in toxicity, thus, loaded diblock polymer micelles containing 5&ndash, 15 wt % free PPO are potential candidates for in vitro and in vivo application as potent MDR inhibitors and drug carriers.

We report novel pH-reversibly surface-shielded polyplexes with enhanced gene transfer activity upon systemic administration. A four-arm-structured sequence-defined cationic oligomer KK[HK[(H-Sph-K)3HC]2]2 was designed and synthesized on solid-phase, containing additional lysine residues not only for improved pDNA polyplex stability, but also providing attachment points for subsequent polyplex functionalization with amine-reactive shielding polymers. Herein, the surface of polyplexes was shielded with hydrophilic polymers, monovalent PEG or monovalent and multivalent pHPMA, optionally attached to the polyplex via the acid-labile linker AzMMMan. Overall, surface modification with PEG or pHPMA resulted in a decrease in the zeta potential of polyplexes, consistent with the degree of surface shielding. At pH 6.0, only polyplexes modified via the acid-labile linkage showed an increase in zeta potential, consistent with a "deshielding" in acidic environment, expected as beneficial for endosomal escape. Shielding was more efficient for multivalent pHPMA (20kDa, 30kDa) as compared to monovalent pHPMA (10kDa, 20kDa, 30kDa) or PEG (5kDa). In vitro transfection studies revealed higher gene expression by the polyplexes with the acid-labile shield as compared to their irreversibly shielded counterparts. Intravenous administration of AzMMMan-pHPMA modified polyplexes in an in vivo tumor mouse model mediated enhanced gene expression in the subcutaneous tumor and reduced undesirable expression in the liver.

Design, controlled synthesis, physico-chemical and biological characteristics of novel well-defined biodegradable star-shaped copolymers intended for advanced drug delivery is described. These new biocompatible star copolymers were synthesised by grafting monodispersed semitelechelic linear (sL) N-(2-hydroxypropyl)methacrylamide copolymers onto a 2,2-bis(hydroxymethyl)propionic acid (bisMPA)-based polyester dendritic core of various structures. The hydrodynamic diameter of the star copolymer biomaterials can be tuned from 13 to 31 nm and could be adjusted to a given purpose by proper selection of the bisMPA dendritic core type and generation and by considering the sL copolymer molecular weight and polymer-to-core molar ratio. The hydrolytic degradation was proved for both the star copolymers containing either dendron or dendrimer core, showing the spontaneous hydrolysis in duration of few weeks. Finally, it was shown that the therapy with the biodegradable star conjugate with attached doxorubicin strongly suppresses the tumour growth in mice and is fully curative in most of the treated animals at dose corresponding approximately to one fourth of maximum tolerated dose (MTD) value. Both new biodegradable systems show superior efficacy and tumour accumulation over the first generation of star copolymers containing non-degradable PAMAM core.

Identifying protein targets of bioactive small molecules often requires complex, lengthy development of affinity probes. We present a method for stochastic modification of small molecules of interest with a photoactivatable phenyldiazirine linker. The resulting isomeric mixture is conjugated to a hydrophilic copolymer decorated with biotin and a fluorophore. We validated this approach using known inhibitors of several medicinally relevant enzymes. At least a portion of the stochastic derivatives retained their binding to the target, enabling target visualization, isolation, and identification. Moreover, the mix of stochastic probes could be separated into fractions and tested for binding affinity. The structure of the active probe could be determined and the probe resynthesized to improve binding efficiency. Our approach can thus enable rapid target isolation, identification, and visualization, while providing information required for subsequent synthesis of an optimized probe.

We developed a new simplified method for the synthesis of well-defined linear, diblock, or starlike N-(2-hydroxypropyl)methacrylamide (HPMA)-based polymer drug carriers using controlled reversible addition-fragmentation chain transfer polymerization. The prepared monodispersed polymers are after the drug attachment intended for enhanced anticancer therapy. This new approach significantly reduces the number of required synthetic steps and minimizes the consumption of organic solvents during the synthesis. As a result, highly defined linear, diblock, and starlike copolymers designed for pH-triggered drug activation/release in tumor tissue were formed in sufficient amounts for further physicochemical and biological studies. Within the synthesis, we also developed a new procedure for the selective deprotection of tert-butoxycarbonyl hydrazide and amine groups on hydrophilic HPMA copolymers, including the one-pot removal of polymer end groups. We studied and described in detail the kinetics and efficacy of the deprotection reaction. We believe the simplified synthetic approach facilitates the preparation of polymer conjugates bound by the pH-sensitive hydrazone bond and their application in tumor treatment.

Anthracyclines, e.g. doxorubicin, pirarubicin, are widely used as cytostatic agents in the polymer nanotherapeutics designed for the highly effective antitumor therapy with reduced side effects. However, their precise dosage scheme needs to be optimized, which requires an accurate method for their quantification on the cellular level in vitro during nanocarrier development and in body fluids and tissues during testing in vivo. Various methods detecting the anthracycline content in biological samples have already been designed. Most of them are highly demanding and they differ in exactness and reproducibility. The cellular uptake and localization is predominantly observed and determined by microscopy techniques, the anthracycline content is usually quantified by chromatographic analysis using fluorescence detection. We reviewed and compared published methods concerning the detection of anthracycline nanocarriers.

High-molecular-weight star polymer drug nanocarriers intended for the treatment and/or visualisation of solid tumours were synthesised, and their physico-chemical and preliminary in vitro biological properties were determined. The water-soluble star polymer carriers were prepared by the grafting of poly(amido amine) (PAMAM) dendrimers by hetero-telechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, synthesised by the controlled radical Reversible Addition Fragmentation chain Transfer (RAFT) polymerisation. The well-defined star copolymers with Mw values ranging from 2 · 10(5) to 6 · 10(5) showing a low dispersity (approximately 1.2) were prepared in a high yield. A model anticancer drug, doxorubicin, was bound to the star polymer through a hydrazone bond, enabling the pH-controlled drug release in the target tumour tissue. The activated polymer arm ends of the star copolymer carrier enable a one-point attachment for the targeting ligands and/or a labelling moiety. In this study, the model TAMRA fluorescent dye was used to prove the feasibility of the polymer carrier visualisation by optical imaging in vitro. The tailor-made structure of the star polymer carriers should facilitate the synthesis of targeted polymer-drug conjugates, even polymer theranostics, for simultaneous tumour drug delivery and imaging.

Proteases are directly involved in cancer pathogenesis. Expression of fibroblast activation protein (FAP) is upregulated in stromal fibroblasts in more than 90% of epithelial cancers and is associated with tumor progression. FAP expression is minimal or absent in most normal adult tissues, suggesting its promise as a target for the diagnosis or treatment of various cancers. Here, we report preparation of a polymer conjugate (an iBody) containing a FAP-specific inhibitor as the targeting ligand. The iBody inhibits both human and mouse FAP with low nanomolar inhibition constants but does not inhibit close FAP homologues dipeptidyl peptidase IV, dipeptidyl peptidase 9, and prolyl oligopeptidase. We demonstrate the applicability of this iBody for the isolation of FAP from cell lysates and blood serum as well as for its detection by ELISA, Western blot, flow cytometry, and confocal microscopy. Our results show the iBody is a useful tool for FAP targeting in vitro and potentially also for specific anticancer drug delivery.

In this study, we describe the design, synthesis, and physicochemical and preliminary biological characteristics of new biodegradable, high-molecular-weight (HMW) drug delivery systems with star-like architectures bearing the cytotoxic drug doxorubicin (DOX) attached by a hydrazone bond-containing spacer. The star polymers were synthesized by grafting semitelechelic N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers on a 2,2-bis(hydroxymethyl)propionic acid- (bis-MPA-) based polyester dendritic core. The molecular weight of the star polymers ranged from 280 to 450 000 g/mol and could be adjusted by proper selection of the bis-MPA dendrimer generation and by considering the polymer to dendrimer molar ratio. The biodegradation of the polymer conjugates is based on the spontaneous slow hydrolysis of the dendritic core in neutral physiological conditions. Hydrazone spacers in the conjugates were fairly stable at neutral pH (7.4) mimicking blood stream conditions, and DOX was released from the conjugates under mild acidic conditions simulating the tumor cell microenvironment in endosomes and lysosomes (pH 5). Finally, we have shown the significant in vitro cytotoxicity of the star polymer-DOX conjugate on selected cancer cell lines with IC50 values almost comparable with that of the free drug and higher than that observed for a linear polymer-DOX conjugate with much lower molecular weight.

The renaissance of phenotypic screening leads to the discovery of biologically active small molecules resulting in a high need to quickly and efficiently identify their protein target. This is most commonly done by a preparation of an affinity probe for the target pull-down, which requires time-consuming structure-activity relationship studies to identify proper position for an attachment of an affinity anchor with necessary linker. We set out to circumvent this step altogether and prepare stochastic conjugates that would enable fast target identification without prior structural information or testing of suitable linker attachment position. We suggest that they could be used also for the identification of unknown targets of phenotypically active compounds as well as off-targets of established active compounds. To test the new approach, we selected several model protein targets – glutamate carboxypeptidase II (GCPII, also known as prostate specific membrane antigen, PSMA) expressed in prostate cancer, fibroblast activation protein (FAP) in tumor stroma, and aspartic protease family, specifically pepsin, cathepsin D and HIV protease. For each, we stochastically modified their corresponding known biologically active small-molecule inhibitors using a linker with a photoactivatable diazirine group. Resulting reaction mixture contained various inhibitor isomers, whose biologic activity was not compromised by the position of the linker attachment as well as those where it was. Afterwards, the reaction mixtures were conjugated to N-(2-hydroxypropyl)methacrylamide copolymers (called iBodies) carrying biotin and a fluorophore, which were used as fully synthetic antibody mimetics. In all cases, stochastic polymer conjugates were able to pull-down the corresponding target protein from cell lysates. For the GCPII conjugate, we then analyzed the pulled-down proteins by mass spectrometry, which clearly identified GCPII as the target protein of the conjugate, even with no prior knowledge needed. For GCPII and FAP, which are cell surface receptors, those same conjugates were able to visualize the proteins on cells both in confocal microscopy and flow cytometry. On a model case of GCPII we decided to identify the position of the “productive” linker attachment so that it would allow rational synthesis for future use. Using HPLC, we separated the reaction mixture into several fractions, each enriched in a specific linker attachment position. As expected, the fractions differed in binding properties compared to the whole mixture. The position of the linker attachment was determined by mass spectrometry for selected active and inactive fractions. The identified linker positions and activity of the compounds corresponded to the known mode of binding of the small molecule into the active site of GCPII. Polymer conjugates prepared from the most favorable fraction had improved binding properties. This “improved” conjugate allowed more efficient isolation and subsequent identification of GCPII by mass spectrometry. These results show that stochastic modification of bioactive small molecule combined with the avidity effect on iBodies allowed us to avoid lengthy structure-activity-relationship studies and enabled identification of protein target and its visualization using a single conjugate. This technology might contribute to fast and facile identification of protein targets of phenotypically active small molecules. This work was supported by Grant No. GA16-02938S from the Grant Agency of the Czech Republic, InterBioMed Project LO 1302 from the Ministry of Education of the Czech Republic, and by the Charles University, project GA UK No. 1510-243-250045. Citation Format: Kristyna Blazkova, Petr Šimon, Tomáš Knedlík, Petra Dvořáková, Anna Březinová, Libor Kostka, Vladimír Šubr, Jan Konvalinka, Pavel Šácha. From bioactive small molecule to an identified protein target: A new method combining stochastic photomodification with a synthetic antibody mimetic [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B147.

In recent years, polymer drug carriers based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers with pH-triggered drug release have shown enhanced uptake in solid tumors and excellent antitumor activity. Here, the impact of the structure of the acid-labile spacer between the drug and the polymer carrier on the biodistribution of both the drug and the carrier was studied using in vivo noninvasive multispectral optical imaging of dual fluorescently labeled HPMA copolymers. Five different spacers containing a pH-sensitive hydrazone bond were synthesized and used to combine a fluorescent model drug with a polymer backbone, conjugated with another non-releasable fluorescent dye. Two copolymers differing in polymer chain structure (linear and star-like) and molecular weight (30 and 200kDa) were used to distinguish between carriers with molecular weights above and below the limit for renal filtration. The rate of model drug release from the conjugates was determined in vitro. The biodistributions of the six most promising conjugates were investigated in vivo in athymic nude mice inoculated with human colon carcinoma xenograft. The structure of the spacer in the vicinity of the hydrazone bond significantly influenced the release rate of the model drug. The slow release rate of a pyridyl group bearing spacer resulted in a greater amount of the model drug being transported to the tumor, which was independent of the carrier structure. The results of this study emphasize the importance of careful selection of the structure and appropriate spacer when designing polymer conjugates intended for passive tumor targeting.

Oncolytic adenoviruses (Ads) selectively replicate within and destroy cancer cells, making them one of the most powerful therapeutics available. However, all robust responses reported to date (1–3) have relied on intratumoral injection, and until Ads can achieve efficacy following intravenous delivery their clinical use will be restricted. In particular, systemic delivery of Ads to treat metastatic cancer will require improved circulation kinetics, extravasation from the bloodstream into the tumor, and intratumoral penetration. After intravenous delivery, Ads bind antibodies, complement, and blood cells (4,5). In response, “stealthing” technologies have been developed, whereby Ads are coated with biocompatible polymers such as polyethylene glycol or N-(2-hydroxypropyl)methacrylamide (6–9). Stealthing removes natural Ad tropism and prevents binding to blood components, leading to extended circulation (10). However, by stealthing Ads to provide sufficient protection during delivery, their ability to infect cells upon arrival in tumors is often compromised. To meet this challenge, polymers can be designed to selectively degrade upon exposure to the tumor milieu. This is possible because disregulated growth of cells within tumors creates a hypoxic and low pH environment that is distinct from normal tissue (11,12). We report the development of a coating polymer that enhances circulation and also allows triggered uncoating and reactivation of Ads within tumors. Despite benefiting from the enhanced permeability retention effect (13), the passage of stealthed Ads from the bloodstream into tumors is still suboptimal, with less than 0.1% of dose achieving deposition in the tumor (4). Furthermore, the intratumoral distribution of Ads remains exclusively perivascular, and without penetration deep into the tumor, the therapeutic effect is restricted to a small proportion of its mass. Such limitations are shared by other therapeutics, such as drug–polymer conjugates and liposomes (14–17). In response, external stimuli have been applied to improve uptake into and spread through tumors (18–21). We feel that focused ultrasound is the safest and most clinically applicable of these strategies. Indeed, our in vitro studies have shown that ultrasound can provide a powerful stimulus to propel Ads deep into tumor-mimicking material (22). To achieve these effects, Ads were coadministered with a microbubble formulation (SonoVue [SV]), which provided nuclei for the initiation of a phenomenon known as inertial cavitation. This term describes the expansion, contraction, and violent collapse of a bubble in response to ultrasound, which creates microstreaming and shock waves. Such events can be used to move macromolecules more than 200 µm (21). The use of microbubbles and ultrasound to improve Ad delivery to tumors is ideal because the ultrasound pressures required are modest and clinically applicable and the inertial cavitation events created are targetable and produce distinct nonharmonic emissions that can be mapped, providing valuable feedback on the success of the procedure (23). We have improved the efficacy of Ads by combining stealthing and ultrasound to overcome their poor pharmacokinetics and limited extravasation and penetration into the tumor.

The synthesis, characterization and results of evaluation of the biological behavior of HPMA copolymer conjugates bearing anti-cancer drugs doxorubicin and mitomycin C are described. Two HPMA copolymer carrier types were synthesized: the linear copolymer and the biodegradable high-molecular-weight diblock copolymer containing a degradable disulfide bond. The polymer-drug conjugates incubated in buffers modeling the intracellular environment released the drugs more rapidly than those incubated in bloodstream conditions. Significant in vitro and in vivo antitumor synergistic activity of the conjugates in the treatment of EL-4 T-cell demonstrates their high potential for solid tumor treatment.

In this work, design and synthesis of high-molecular-weight N-(2-hydroxypropyl)methacrylamide-based polymer drug delivery systems tailored for cancer therapy is summarized. Moreover, the influence of their architecture on tumor accumulation and in vivo anti-cancer efficacy is discussed. Mainly, the high-molecular-weight delivery systems, such as branched, grafted, multi-block, star-like or micellar systems, with molecular weights greater than the renal threshold are discussed and reviewed in detail.

New amphiphilic diblock polymer nanotherapeutics serving simultaneously as a drug delivery system and an inhibitor of multidrug resistance were designed, synthesized, and evaluated for their physico-chemical and biological characteristics. The amphiphilic character of the diblock polymer, containing a hydrophilic block based on the N-(2-hydroxypropyl)methacrylamide copolymer and a hydrophobic poly(propylene oxide) block (PPO), caused self-assembly into polymer micelles with an increased hydrodynamic radius (Rh of approximately 15nm) in aqueous solutions. Doxorubicin (Dox), as a cytostatic drug, was bound to the diblock polymer through a pH-sensitive hydrazone bond, enabling prolonged circulation in blood, the delivery of Dox into a solid tumor and the subsequent stimuli-sensitive controlled release within the tumor mass and tumor cells at a decreased pH. The applicability of micellar nanotherapeutics as drug carriers was confirmed by an in vivo evaluation using EL4 lymphoma-bearing C57BL/6 mice. We observed significantly higher accumulation of micellar conjugates in a solid tumor because of the EPR effect compared with similar polymer-drug conjugates that do not form micellar structures or with the parent free drug. In addition, highly increased anti-tumor efficacy of the micellar polymer nanotherapeutics, even at a sub-optimal dose, was observed. The presence of PPO in the structure of the diblock polymer ensured, during in vitro tests on human and mouse drug-sensitive and resistant cancer cell lines, the inhibition of P-glycoprotein, one of the most frequently expressed ATP-dependent efflux pump that causes multidrug resistance. In addition, we observed highly increased rate of the uptake of the diblock polymer nanotherapeutics within the cells. We suppose that combination of unique properties based on MDR inhibition, stimuli sensitiveness (pH sensitive activation of drug), improved pharmacokinetics and increased uptake into the cells made the described polymer micelle a good candidate for investigation as potential drug delivery system.

The enzymatic release of a model drug from a polymer carrier inside a tumor using multispectral optical imaging in vivo in nude mice bearing colorectal carcinomas HT-29 and DLD-1 is demonstrated. Much higher release rate in vivo from a linear (30 kDa) (N-2-hydroxypropyl)methacrylamide-based polymer compared with a high molecular weight branched (170 kDa) polymer conjugate is observed, probably due to steric hindrance of the cleavable spacer of the latter polymer to proteolytic enzymes. There is no significant difference in the relative biodistribution of the two polymers, but the branched polymer circulates much longer. Both polymers are labeled with two different fluorophores. Dyomics-676 as a drug model is attached to the polymer via an enzymatically cleavable Gly-Phe-Leu-Gly spacer; Dyomics 782 is bound to the same polymer via a nondegradable amide bond, enabling the tracking of the polymer carrier after i.v. application to mice.

Various biodegradable micellar polymer drug carriers based on N-(2-hydroxypropyl)methacrylamide copolymers and their conjugates with doxorubicin are synthesized. The conjugates are designed to treat solid tumors and subsequently to allow carrier elimination from the body. All copolymers self-assemble in aqueous solution into supramolecular structures with highly hydrophobic cholesterol derivatives in the core and doxorubicin located in the hydrophilic shell. The drug is released quickly from the micelles incubated at pH = 5.0 (modelling the endosomes of tumor cells). Slower release of cholesterol derivatives leading to disintegration of the micelles is observed.

To assist in overcoming the inherent instability of nucleic acid-containing polyplexes in physiological solutions, we have here set out to develop removable nanocoatings for modifying the surface of siRNA-based nanoparticles. Here, N-(2-hydroxypropyl)methacrylamide (HPMA) based copolymers containing carbonylthiazolidine-2-thione (TT) reactive groups in their side chains bound via disulfide spacers to the polymeric backbone were synthesized, and these copolymers were used to coat the surface of polyplexes formed by the self-assembly of anti-Luciferase siRNA with the polycations polyethylene imine (PEI) and poly(HPMA)-grafted poly(l-lysine) (GPL). The coating process was monitored by analyzing changes in the weight-average molecular weight (M(w)), the hydrodynamic radius (R(h)), and the zeta-potential (ζ) of the polyplexes, using both static (SLS) and dynamic (DLS) light scattering methods. The outlined methods resulted in the attachment of, on average, 28 polymer molecules to the surface of the polyplexes, forming a ∼5-nm-thick hydrophilic stealth coating. Initial efforts to develop RGD-targeted coated polyplexes are also described. Atomic force microscopy (AFM) showed an angular polyplex structure and confirmed the narrow size distribution of the coated nanoparticles. The stability of the polymer-coated and uncoated polyplexes was evaluated by gel electrophoresis and by turbidity measurements, and it was found that modifying the surface of the siRNA-containing polyplexes substantially improved their stability in physiological solutions. Using polymer-coated GPL-based polyplexes containing anti-Luciferase siRNA, we finally also obtained some initial proof-of-principle for time- and concentration-dependent target-specific gene silencing, suggesting that these systems hold significant potential for further in vitro and in vivo evaluation.

Short polypeptides with four pentad repeats, (VPGVG)(4) and (VPAVG)(4), were synthesised by manual fluorenylmethoxycarbonyl/tert-butyl (Fmoc/t-Bu) solid phase peptide synthesis using a convergent approach. In the next step, the peptides were coupled via their N-terminus with activated semi-telechelic poly(ethylene glycol) O-(N-Fmoc-2-aminoethyl)-O'-(2-carboxyethyl)undeca(ethylene glycol) (Fmoc-PEG-COOH) to yield monodisperse Fmoc-PEG-peptide diblock copolymer. Both the presence of the terminal hydrophobic Fmoc group and the hydrophilic PEG chain in the copolymers were shown to play a crucial role in their self-associative behaviour, leading to reversible formation of supramolecular thermoresponsive assemblies. The peptides and their PEG derivatives were characterised by HPLC, NMR and MALDI-TOF MS. The associative behaviour of the peptides and their PEG derivatives was studied by dynamic light scattering, MAS NMR and phase contrast microscopy. [image: see text]

Efficient intravenous delivery is the greatest single hurdle, with most nanotherapeutics frequently found to be unstable in the harsh conditions of the bloodstream. In the case of nanotherapeutics for gene delivery, viral vectors are often avidly recognized by both the innate and the adaptive immune systems. So, most modern delivery systems have benefited from being coated with hydrophilic polymers. Self-assembling delivery systems can achieve both steric and lateral stabilization following surface coating, endowing them with much improved systemic circulation properties and better access to disseminated targets; similarly, gene delivery viral vectors can be ‘stealthed’ and their physical properties modulated by surface coating. Polymers that start degrading under acidic conditions are increasingly investigated as a pathway to trigger the release of drugs or genes once the carrier reaches a slightly acidic tumor environment or after the carrier has been taken up by cells, resulting in the localization of the polymer in acidic endosomes and lysosomes. Advances in the design of acid-degradable drug and gene delivery systems have been focused and discussed in this article with stress placed on HPMA-based copolymers. We designed a system that is able to “throw away” the polymer coat after successful transport of the vector into a target cell. Initial biological studies were performed and it was demonstrated that this principle is applicable for real adenoviral vectors. It was shown that the transfection ability of coated virus at pH 7.4 is 75 times lower then transfection at pH 5.4.

A series of model linear copolymers of 2-hydroxyethyl methacrylate (HEMA) and a sterically hindered amine derivative [N-(2,2,6,6-tetramethyl-piperidin-4-yl)methacrylamide (HAS)] were synthesized and characterized. Scavenging activities of the copolymers against reactive oxygen species (peroxyl and hydroxyl radicals) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals were determined. It was found that copolymers with medium HAS content (3.5-4.0 mol %) were better scavengers than copolymers with lower and higher HAS content and also than polyHEMA and polyHAS homopolymers and the HAS monomer. Importantly, these copolymers compared favorably even to established low-molecular weight antioxidant standards (BHA and dexpanthenol). Monomer reactivity ratios were determined, and the microstructure of the copolymers was assessed. Subsequently, cross-linked copolymers in the powder and film forms with optimal HAS content were synthesized. Their scavenging activities against the three types of radicals were determined, revealing that these hydrogels are potent scavengers of reactive oxygen species.

Adenovirus type 5 (Ad5) gene therapy vectors require protection against antibodies, complement proteins and blood cells if they are to be delivered intravenously to treat metastatic disease. Such protection can be achieved by chemically modifying Ad5 with polymers based on hydrophilic HPMA. Here, such polymers were designed to include side chains bearing reactive carbonyl thiazolidine-2-thione groups (TTs) to covalently modify available amino groups of the lysine residues in the Ad5 capsid. Furthermore, the inclusion of side chains bearing positively charged quaternary ammonium groups (QAs) was designed to improve electrostatic interaction of the polymers with negatively charged Ad5 hexon protein. Finally, to enable triggered uncoating and reactivation of the Ad5, either the TTs or both the TTs and the QAs were linked to polymer backbone via reductively degradable disulfide bonds. SDS-PAGE demonstrated that these polymers covalently modified Ad5 capsid proteins in a reduction reversible manner. In infection studies, polymers containing QAs prevented binding of coagulation factor X to Ad5. Furthermore, the antibody and complement mediated binding of Ad5 to erythrocytes was reduced by such polymers (>95% without polymer, 25% following coating). These data indicate that coating Ad5 therapeutics with such polymers will improve blood circulation half-life and deposition at disease sites.

Vesicles bearing either cationic (amino) groups or zwitterionic (amino acid) groups on the surface were coated with a reactive multivalent hydrophilic N-(2-hydroxypropyl)methacrylamide polymer (PHPMA) and its positively charged analogue (3 mol % quaternary ammonium groups), both having reactive thiazolidine-2-thione (TT) groups randomly distributed along the polymer chain. The vesicles were dispersed in water at a concentration of 1 mg/mL. The effect of surface charges of model vesicles on the surface coating efficiency was evaluated. The changes in the weight-average molecular weight, in the hydrodynamic size, and in the zeta-potential of model vesicles were tested using light scattering methods. The most effective coating of vesicles was observed for the zwitterionic vesicles coated with the positively charged hydrophilic PHPMA-TT copolymer at a concentration of reactive polymer cp = 2 mg/mL. The coating efficiency was more than 1 order of magnitude higher than that obtained for positively charged vesicles coated by the uncharged hydrophilic polymer at the same cp.