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The mouse Nrampl (Bcg) gene on chromosome 1 exerts pleiotropk effects on macrophage function. The gene is known to affect presentation of mycobacteria, and other antigens in vitro, so that macrophages carrying the resistant Bcg allele better support the proliferation of antigen-specific T cells compared with macrophages of the sensitive phenotype. To determine whether the Bcg allele could affect in vivo the antibody response to antigens not related to mycobacterial infections, we tested the primary and secondary responses to sheep red blood cells (SRBC) and glycosylated bovine insulin (G-insulin) in two pairs of Bcg congenic strains: BALB/c (Bcgs) versus BALB/c.CD2 (Bcgr), and B10.A (Bcgs) versus B10Ar (Bcgr), and in C57BL/10ScSn (B10; Bcgs) and A/J (Bcgr) mict. Furthermore, the antigen-specific proliferative responses of T cells primed in vivo by protein antigens were also tested in Bcg congenic mice. We found no significant difference in vim antibody response either to SRBC or G-insulin between the Bcgr and Bcgs strains. The magnitude of in vitro antigen-specific proliferation of lymph node cells sensitized in vivo by hen egg lysozyme (HEL) or chicken ovalbumin (OVA) was also similar in Bcgs and Bcgr congenic mice. However, we have documented a higher antigen-presenting capacity of Bcgr macrophages in vitro antigen-specific proliferation to OVA. Since the macrophages are the only cells in which the Nrampl gene is expressed, we suggest that the activity of other types of antigen-presenting cells masks the effect of the Bcgr allele on antigen-presentation in vivo. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Declaration of Competing Interest Weibo Cai is a scientific advisor, stockholder, and grantee of Focus-X Therapeutics, Inc. All other authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Competing Interests: Declaration of competing interest The authors declare no conflict of interest.

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.

Galectin-3 (Gal-3) participates in many cancer-related metabolic processes. The inhibition of overexpressed Gal-3 by, e.g., β-galactoside-derived inhibitors is hence promising for cancer treatment. The multivalent presentation of such inhibitors on a suitable biocompatible carrier can enhance the overall affinity to Gal-3 and favorably modify the interaction with Gal-3-overexpressing cells. We synthesized a library of C-3 aryl-substituted thiodigalactoside inhibitors and their multivalent N -(2-hydroxypropyl)methacrylamide (HPMA)-based counterparts with two different glycomimetic contents. Glycopolymers with a higher content of glycomimetic exhibited a higher affinity to Gal-3 as assessed by ELISA and biolayer interferometry. Among them, four candidates (with 4-acetophenyl, 4-cyanophenyl, 4-fluorophenyl, and thiophen-3-yl substitution) were selected for further evaluation in cancer-related experiments in cell cultures. These glycopolymers inhibited Gal-3-induced processes in cancer cells. The cyanophenyl-substituted glycopolymer exhibited the strongest antiproliferative, antimigratory, antiangiogenic, and immunoprotective properties. The prepared glycopolymers appear to be prospective modulators of the tumor microenvironment applicable in the therapy of Gal-3-associated cancers.

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 (IC 50  ~ 2.3-17.4 μM) and six human (IC 50  ~ 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The study describes the synthesis, physicochemical properties, and biological evaluation of polymer therapeutics based on N -(2-hydroxypropyl)methacrylamide (HPMA) copolymers intended for a tumor-targeted immuno-oncotherapy. Water-soluble linear and cholesterol-containing HPMA precursors were synthesized using controlled reversible addition-fragmentation chain transfer polymerization to reach molecular weight M n about 2 × 10 4 g·mol -1 and low dispersity. These linear or self-assembled micellar conjugates, containing immunomodulatory agent cucurbitacin-D (CuD) or the anticancer drug doxorubicin (Dox) covalently bound by the hydrolytically degradable hydrazone bond, showed a hydrodynamic size of 10-30 nm in aqueous solutions. The CuD-containing conjugates were stable in conditions mimicking blood. Importantly, a massive release of active CuD in buffer mimicking the acidic tumor environment was observed. In vitro, both the linear (LP-CuD) and the micellar (MP-CuD) conjugates carrying CuD showed cytostatic/cytotoxic activity against several cancer cell lines. In a murine metastatic and difficult-to-treat 4T1 mammary carcinoma, only LP-CuD showed an anticancer effect. Indeed, the co-treatment with Dox-containing micellar polymer conjugate and LP-CuD showed potentiation of the anticancer effect. The results indicate that the binding of CuD, characterized by prominent hydrophobic nature and low bioavailability, to the polymer carrier allows a safe and effective delivery. Therefore, the conjugate could serve as a potential component of immuno-oncotherapy schemes within the next preclinical evaluation.

The study compared the physico-chemical and biological properties of a water-soluble star-like polymer nanomedicine with three micellar nanomedicines formed by self-assembly of amphiphilic copolymers differing in their hydrophobic part (statistical, block and thermosensitive block copolymers). All nanomedicines showed a pH-responsive release of the drug, independent on polymer structure. Significant penetration of all polymer nanomedicines into tumor cells in vitro was demonstrated, where the most pronounced effect was observed for statistical- or diblock copolymer-based micellar systems. Tumor accumulation in vivo was dependent on the stability of the nanomedicines in solution, being the highest for the star-like system, followed by the most stable micellar nanomedicines. The star-like polymer nanomedicine showed a superior therapeutic effect. Since the micellar systems exhibited slightly lower systemic toxicity, they may exhibit the same efficacy as the star-like soluble system when administered at equitoxic doses. In conclusion, treatment efficacy of studied nanomedicines was directly controlled by the drug pharmacokinetics, namely by their ability to circulate in the bloodstream for the time needed for effective accumulation in the tumor due to the enhanced permeability and retention (EPR) effect. Easy and scalable synthesis together with the direct reconstitution possibility for nanomedicine application made these nanomedicines excellent candidates for further clinical evaluation., Competing Interests: Declaration of Competing Interest The authors declare none conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 5-40 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 5-15 wt % free PPO are potential candidates for in vitro and in vivo application as potent MDR inhibitors and drug carriers., Competing Interests: The authors declare no conflict of interest.

Herein, the biodegradable micelle-forming amphiphilic N-(2-hydroxypropyl) methacrylamide (HPMA)-based polymer conjugates with the anticancer drug doxorubicin (Dox) designed for enhanced tumor accumulation were investigated, and the influence of their stability in the bloodstream on biodistribution, namely, tumor uptake, and in vivo antitumor efficacy were evaluated in detail. Dox was attached to the polymer carrier by a hydrazone bond enabling pH-controlled drug release. While the polymer-drug conjugates were stable in a buffer at pH 7.4 (mimicking bloodstream environment), Dox was released in a buffer under mild acidic conditions modeling the tumor microenvironment or cells. The amphiphilic polymer carriers differed in the structure of hydrophobic cholesterol derivative moieties bound to the HPMA copolymers via a hydrolyzable hydrazone bond, exhibiting different rates of micellar structure disintegration at various pH values. Considerable dependence of the studied polymer-drug conjugate biodistribution on the stability of the micellar structure was observed in neutral, bloodstream-mimicking, environment, showing that a faster rate of the micelle disintegration in pH 7.4 increased the conjugate blood clearance, decreased tumor accumulation, and significantly reduced the tumor:blood and tumor:muscle ratios. Similarly, the final therapeutic outcome was strongly affected by the stability of the micellar structure because the most stable micellar conjugate showed an almost similar therapeutic outcome as the water-soluble, nondegradable, high-molecular-weight starlike HPMA copolymer-Dox conjugate, which was highly efficient in the treatment of solid tumors in mice. Based on the results, we conclude that the bloodstream stability of micellar polymer-anticancer drug conjugates, in addition to their low side toxicity, is a crucial parameter for their efficient solid tumor accumulation and high in vivo antitumor activity.

Polymer carriers based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers with incorporated organic nitrates as nitric oxide (NO) donors were designed with the aim to localise NO generation in solid tumours, thus highly increasing the enhanced permeability and retention (EPR) effect. The NO donors were coupled to the polymer carrier either through a stable bond or through a hydrolytically degradable, pH sensitive, bond. In vivo, the co-administration of the polymer NO donor and HPMA copolymer-bound cytotoxic drug (doxorubicin; Dox) resulted in an improvement in the treatment of murine EL4 T-cell lymphoma. The polymer NO donors neither potentiated the in vitro toxicity of the cytotoxic drug nor exerted any effect on in vivo model without the EPR effect, such as BCL1 leukaemia. Thus, an increase in passive accumulation of the nanomedicine carrying cytotoxic drug via NO-enhanced EPR effect was the operative mechanism of action. The most significant improvement in the therapy was observed in a combination treatment with such a polymer conjugate of Dox, which is characterised by increased circulation in the blood and efficient accumulation in solid tumours. Notably, the combination treatment enabled the development of an anti-tumour immune response, which was previously demonstrated as an important feature of HPMA-based polymer cytotoxic drugs.

The tumor-specific targeting of cancerostatics using polymer drug carriers represents a potential strategy to achieve an effective treatment with reduced side toxicity. Synthetic water-soluble copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA) are carriers with tunable architecture and drug loading, tumor-specific accumulation of the drug, and its controlled release. We describe a combination treatment of murine EL4 T cell lymphoma with HPMA-based star conjugates (M w 250,000gmol -1 ) of doxorubicin (Dox) or docetaxel (Dtx) designed for enhanced tumor accumulation and combination therapy. Although the combination of linear conjugates (M w =28,000gmol -1 ) containing Dox or Dtx resulted in an additive effect in the treatment of the lymphoma, the opposite was observed in the combination of two star conjugates with Dox or Dtx, as the star Dtx conjugate decreased the treatment efficacy of the star Dox conjugate. The Dtx conjugate alone was virtually ineffective in the reduction of tumor growth or survival time extension; thus, a curative effect could be solely attributed to the Dox-containing conjugate. When Dtx was delivered to the tumor on the same polymer carrier as Dox, the efficacy of the Dox-induced treatment was reduced to a lesser extent. No reduction was found when Dtx was delivered by a linear polymer or applied as a free drug. The phenomenon was strictly related to the enhanced permeability and retention (EPR) effect, as it was not observed in BCL1 leukemia, a model without EPR. The diminished treatment outcome in the combination therapy with the two star conjugates was underlined by the significantly decreased accumulation of Dox in the tumor. The use of the drug-free polymer carrier instead of the Dtx-containing star conjugate did not reduce the treatment efficacy of the Dox conjugate. Thus, the physicochemical characteristics of the polymer carrier designed for tumor-specific drug delivery systems control the activity of the respective drug, leading to changes within the tumor microenvironment that can determine ultimate efficacy of the combination therapy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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 (R h 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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We have shown previously that a short pentapeptide derived from the mycobacterial heat shock protein hsp65 can be highly activating for the immune system based on its strong reactivity with the early activation antigen of lymphocytes CD69. Here, we investigated an optimal form of presentation of this antigen to the cells of the immune system. Four different forms of the dimerized heptapeptide LELTEGY, and of the control inactive dimerized heptapeptide LELLEGY that both contained an extra UV active glycine-tyrosine sequence, were prepared using dihydroxysuccinimidyl oxalate (DSO), dihydroxysuccinimidyl tartarate (DST), dihydroxysuccinimidyl glutarate (DSG), and dihydroxysuccinimidyl suberate (DSS), respectively. Heptapeptides dimerized through DST and DSG linkers had optimal activity in CD69 precipitation assay. Moreover, dimerization of active heptapeptide resulted in a remarkable increase in its proliferation activity and production of cytokines in vitro. Furthermore, while DST and DSG dimerized heptapeptides both significantly enhanced the cytotoxicity of natural killer cells in vitro, only the DSG dimerized compound was active in suppressing growth of melanoma tumors in mice and in enhancing the cytotoxic activity of tumor infiltrating lymphocytes ex vivo. Thus, while the dimerization of the immunoactive peptide caused a dramatic increase in its immunoactivating properties, its in vivo anticancer properties were influenced by the chemical nature of linker used for its dimerization.

We have investigated the effects of low-frequency pulsed electromagnetic field (LF-EMF) produced by BEMER device on experimental mouse T-cell lymphoma EL4 growing on conventional and/or athymic (nude) mice. Exposure to EMF-BEMER slowed down the growth of tumor mass and prolonged the survival of experimental animals. The effect was more pronounced in immuno-compromised nude mice compared to conventional ones. Acceleration of tumor growth was never observed. No measurable levels of Hsp 70 or increased levels of specific anti-EL4 antibodies were detected in the serum taken from experimental mice before and at different intervals during the experiment, i.e. before solid tumor appeared, at the time of its aggressive growth, and at the terminal stage of the disease. A significant synergizing antitumor effect was seen when EL4 tumor-bearing mice were simultaneously exposed to EMF-BEMER and treated with suboptimal dose of synthetic HPMA copolymer-based doxorubicin, DOX(HYD)-HPMA. Such a combination may be especially useful for heavily treated patients suffering from advanced tumor and requiring additional aggressive chemotherapy which, however, at that time could represent almost life-threatening way of medication.