Therapeutic activity and biodistribution of a nano-sized polymer-dexamethasone conjugate intended for the targeted treatment of rheumatoid arthritis.

Rheumatoid arthritis is a chronic inflammatory autoimmune disease caused by alteration of the immune system. Current therapies have several limitations and the use of nanomedicines represents a promising strategy to overcome them. By employing a mouse model of adjuvant induced arthritis, we aimed to evaluate the biodistribution and therapeutic effects of glucocorticoid dexamethasone conjugated to a nanocarrier based on biocompatible N-(2-hydroxypropyl) methacrylamide copolymers. We observed an increased accumulation of dexamethasone polymer nanomedicines in the arthritic mouse paw using non-invasive fluorescent in vivo imaging and confirmed it by the analysis of tissue homogenates. The dexamethasone conjugate exhibited a dose-dependent healing effect on arthritis and an improved therapeutic outcome compared to free dexamethasone. Particularly, significant reduction of accumulation of RA mediator RANKL was observed. Overall, our data suggest that the conjugation of dexamethasone to a polymer nanocarrier by means of stimuli-sensitive spacer is suitable strategy for improving rheumatoid arthritis therapy.

Dexamethasone nanomedicines with optimized drug release kinetics tailored for treatment of site-specific rheumatic musculoskeletal diseases.

The application of polymer-based drug delivery systems is advantageous for improved pharmacokinetics, controlled drug release, and decreased side effects of therapeutics for inflammatory disease. Herein, we describe the synthesis and characterization of linear N-(2-hydroxypropyl)methacrylamide-based polymer conjugates designed for controlled release of the anti-inflammatory drug dexamethasone through pH-sensitive bonds. The tailored release rates were achieved by modifying DEX with four oxo-acids introducing reactive oxo groups to the DEX derivatives. Refinement of reaction conditions yielded four well-defined polymer conjugates with varied release profiles which were more pronounced at the lower pH in cell lysosomes. In vitro evaluations in murine peritoneal macrophages, human synovial fibroblasts, and human peripheral blood mononuclear cells demonstrated that neither drug derivatization nor polymer conjugation affected cytotoxicity or anti-inflammatory properties. Subsequent in vivo tests using a murine arthritis model validated the superior anti-inflammatory efficacy of the prepared DEX-bearing conjugates with lower release rates. These nanomedicines showed much higher therapeutic activity compared to the faster release systems or DEX itself.

Using surface plasmon resonance, capillary electrophoresis and diffusion-ordered NMR spectroscopy to study drug release kinetics.

Nanomedicines, including polymer nanocarriers with controlled drug release, are considered next-generation therapeutics with advanced therapeutic properties and reduced side effects. To develop safe and efficient nanomedicines, it is crucial to precisely determine the drug release kinetics. Herein, we present application of analytical methods, i.e., surface plasmon resonance biosensor technology (SPR), capillary electrophoresis, and 1H diffusion-ordered nuclear magnetic resonance spectroscopy, which were innovatively applied for drug release determination. The methods were optimised to quantify the pH-triggered release of three structurally different drugs from a polymer carrier. The suitability of these methods for drug release characterisation was evaluated and compared using several parameters including applicability for diverse samples, the biological relevance of the experimental setup, method complexity, and the analysis outcome. The SPR method was the most universal method for the evaluation of diverse drug molecule release allowing continuous observation in the flow-through setting and requiring a small amount of sample.

Evaluation of linear versus star-like polymer anti-cancer nanomedicines in mouse models.

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.

The transmission and toxicity of polymer-bound doxorubicin-containing exosomes derived from human adenocarcinoma cells.

Background: Exosomes are extracellular vesicles with the ability to encapsulate bioactive molecules, such as therapeutics. This study identified a new exosome mediated route of doxorubicin and poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA)-bound doxorubicin trafficking in the tumor mass. Materials & methods: Exosome loading was achieved via incubation of the therapeutics with an adherent human breast adenocarcinoma cell line and its derived spheroids. Exosomes were characterized using HPLC, nanoparticle tracking analysis (NTA) and western blotting. Results: The therapeutics were successfully loaded into exosomes. Spheroids secreted significantly more exosomes than adherent cells and showed decreased viability after treatment with therapeutic-loaded exosomes, which confirmed successful transmission. Conclusion: To the best of our knowledge, this study provides the first evidence of pHPMA-drug conjugate secretion by extracellular vesicles.

Background: In cancer treatment, low-molecular-weight drugs (e.g., doxorubicin [DOX]) with a broad spectrum of side effects are commonly used. Through their conjugation with hydrophilic polymers ­ N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers ­ for example, most of the side effects can be reduced. These drug­polymer conjugates are delivered via bloodstream into the tumor. This study aimed to identify a new exosome-mediated route of DOX and polyHPMA(pHPMA)­DOX conjugates trafficking inside the tumor mass. Exosomes are small lipid membrane vesicles constitutively released from most of the cell types, including the tumor cells. Exosomes are able to encapsulate low-molecular-weight drugs. Methods: Exosomes were loaded with DOX and pHPMA-DOX in vitro via coincubation with cancer cells. Exosomes were isolated from the conditioned-cultivation medium after their release from cells and characterized (size, numbers, protein marker profiles). Results: The therapeutics were successfully loaded into exosomes and transmitted to the tumor cells. To the best of our knowledge, this is the first evidence of the pHPMA­drug conjugate secretion by exosomes.

Polymer-ritonavir derivate nanomedicine with pH-sensitive activation possesses potent anti-tumor activity in vivo via inhibition of proteasome and STAT3 signaling.

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.

Coating Persistent Luminescence Nanoparticles With Hydrophilic Polymers for in vivo Imaging.

Persistent luminescence nanoparticles (PLNPs) are innovative nanomaterials highly useful for bioimaging applications. Indeed, due to their particular optical properties, i.e., the ability to store the excitation energy before slowly releasing it for a prolonged period of time, they allow in vivo imaging without auto-fluorescence and with a high target to background ratio. However, as for most nanoparticles (NPs), without any special surface coating, they are rapidly opsonized and captured by the liver after systemic injection into small animals. To overcome this issue and prolong nanoparticle circulation in the bloodstream, a new stealth strategy was developed by covering their surface with poly(N-2-hydroxypropyl)methacrylamide (pHPMA), a highly hydrophilic polymer widely used in nanomedicine. Preliminary in vivo imaging results demonstrated the possibility of pHPMA as an alternative strategy to cover ZnGa2O4:Cr NPs to delay their capture by the liver, thereby providing a new perspective for the formulation of stealth NPs.

Polymer Nanomedicines with Ph-Sensitive Release of Dexamethasone for the Localized Treatment of Inflammation.

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 × 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.

HPMA copolymer-antibody constructs in neoplastic treatment: an overview of therapeutics, targeted diagnostics, and drug-free systems.

In the past decades, nanosized drug delivery systems based on N-(2-hydroxypropyl)methacrylamide copolymers (pHPMA) have gained increasing attention in nanomedicine field due to their hydrophilicity, versatility, biocompatibility, non-toxicity, and non-immunogenicity. Indeed, pHPMA nanosystems with various controlled drug release capabilities inside targeted tissues or cells have been intensively studied. This paper summarizes recent advances in the design and application of pHPMA conjugates with specific antibodies or their fragments, focusing predominantly on the systems for the cancer therapy, particularly, the mechanisms of action of therapeutic antibodies, the approaches of their modification and subsequent attachment of pHPMA and their conjugates with diverse active moieties. Finally, we highlight the major biomedical applications of these antibody-polymer-drug conjugates and consider directions of possible development over the coming decade.

Highly effective anti-tumor nanomedicines based on HPMA copolymer conjugates with pirarubicin prepared by controlled RAFT polymerization.

Here, we describe innovative synthesis of well-defined biocompatible N-(2-hydroxypropyl) methacrylamide (HPMA)-based polymer carriers and their drug conjugates with pirarubicin intended for controlled drug delivery and pH-triggered drug activation in tumor tissue. Polymer carrier synthesis was optimized to obtain well-defined linear HPMA-based polymer precursor with dispersity close to 1 and molar mass close to renal threshold with minimal synthesis steps. The developed synthesis enables preparation of tailored polymer nanomedicines with highly enhanced biological behavior in vivo, especially the biodistribution, urine elimination, tumor accumulation and anticancer activity. STATEMENT OF SIGNIFICANCE: The manuscript reports on novel synthesis and detailed physicochemical characterization and in vivo evaluation of well-defined biocompatible hydrophilic copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA) and their drug conjugates with pirarubicin enabling controlled drug delivery and pH-triggered drug activation in tumor tissue. Polymer carrier synthesis was optimized to obtain well-defined linear HPMA-based polymer precursor with minimal synthesis steps using controlled polymerization. Compared to previously published HPMA-based polymer drug conjugates whose polymer carriers were prepared by classical route via free radical polymerization, the newly prepared polymer drug conjugates exhibited enhanced biological behavior in vivo, especially the prolonged blood circulation, urine elimination, tumor accumulation and excellent anticancer activity. We believe that the newly prepared well-defined polymer conjugates could significantly enhance tumor therapy in humans.

Structure-to-Efficacy Relationship of HPMA-Based Nanomedicines: The Tumor Spheroid Penetration Study.

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.