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.

• Klíčová slova
• AzMMMan, Non-viral gene delivery, PEG, Plasmid DNA, pH-sensitive shielding, pHPMA,
• MeSH
• amidy chemie   MeSH
• DNA chemie   MeSH
• methakryláty chemie   MeSH
• myši MeSH
• technika přenosu genů * MeSH
• techniky in vitro MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• amidy MeSH
• DNA MeSH
• hydroxypropyl methacrylate MeSH Prohlížeč
• methakryláty MeSH

The systemic application of highly potent drugs such as cytostatics poses the risks of side effects, which could be reduced by using a carrier system able to specifically deliver the encapsulated drug to the target tissue. Essential components of a nanoparticle-based drug delivery system include the drug carrier itself, a targeting moiety, and a surface coating that minimizes recognition by the immune system. The present work reports on the preparation, in vitro characterization and in vivo testing of a new delivery system consisting of fluorescent silica nanoparticles functionalised with a non-immunogenic stealth polymer poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA) and a monoclonal antibody IgG M75 that specifically binds to Carbonic Anhydrase IX (CA IX). CA IX is a promising therapeutic target, as it is a hallmark of several hypoxic tumours including colorectal carcinoma. Uniquely in this work, the monoclonal antibody was covalently coupled to the surface of fluorescently labelled silica nanoparticles via a multivalent amino-reactive co-polymer rather than a traditional bivalent linker. The pHPMA-M75 functionalised SiO2 nanoparticles exhibited excellent colloidal stability in physiological media. Their in vitro characterisation by flow cytometry proved a highly specific interaction with colorectal carcinoma cells HT-29. In vivo study on athymic NU/NU nude mice revealed that the SiO2-pHPMA-M75 nanoparticles are capable of circulating in the blood after intravenous administration and accumulate in the tumour at tenfold higher concentration than nanoparticles without specific targeting, with a considerably longer retention time. Additionally, it was found that by reducing the dose administered in vivo, the selectivity of the nanoparticle biodistribution could be further enhanced in favour of the tumour.

• Publikační typ
• časopisecké články MeSH

Nanoparticles (NPs) that form by self-assembly of amphiphilic poly(N-(2-hydroxypropyl)-methacrylamide) (pHPMA) copolymers bearing cholesterol side groups are potential drug carriers for solid tumor treatment. Here, we investigate their behavior in solutions of human serum albumin (HSA) in phosphate buffered saline. Mixed solutions of NPs, from polymer conjugates with or without the anticancer drug doxorubicin (Dox) bound to them, and HSA at concentrations up to the physiological value are characterized by synchrotron small-angle X-ray scattering and isothermal titration calorimetry. When Dox is absent, a small amount of HSA molecules bind to the cholesterol groups that form the core of the NPs by diffusing through the loose pHPMA shell or get caught in meshes formed by the pHPMA chains. These interactions are strongly hindered by the presence of Dox, which is distributed in the pHPMA shell, meaning that the delivery of Dox by the NPs in the human body is not affected by the presence of HSA.

• MeSH
• cholesterol chemie   farmakokinetika   MeSH
• doxorubicin chemie   farmakokinetika   MeSH
• kyseliny polymethakrylové chemie   farmakokinetika   MeSH
• lékové transportní systémy metody   MeSH
• lidé MeSH
• lidský sérový albumin chemie   farmakokinetika   MeSH
• nádory farmakoterapie   metabolismus   MeSH
• nanočástice chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholesterol MeSH
• doxorubicin MeSH
• Duxon MeSH Prohlížeč
• kyseliny polymethakrylové MeSH
• lidský sérový albumin MeSH

Amphiphilic poly( N-(2-hydroxypropyl)methacrylamide) copolymers ( pHPMA) bearing cholesterol side groups in phosphate buffer saline self-assemble into nanoparticles (NPs) which can be used as tumor-targeted drug carriers. It was previously shown by us that human serum albumin (HSA) interacts weakly with the NPs. However, the mechanism of this binding could not be resolved due to overlapping of signals from the complex system. Here, we use fluorescence labeling to distinguish the components and to characterize the binding: On the one hand, a fluorescent dye was attached to pHPMA, so that the diffusion behavior of the NPs could be studied in the presence of HSA using fluorescence lifetime correlation spectroscopy. On the other hand, quenching of the intrinsic fluorescence of HSA revealed the origin of the binding, which is mainly the complexation between HSA and cholesterol side groups. Furthermore, a binding constant was obtained.

• MeSH
• fluorescenční spektrometrie * MeSH
• lidé MeSH
• lidský sérový albumin * metabolismus   MeSH
• makromolekulární látky MeSH
• nanočástice chemie   MeSH
• nosiče léků chemie   MeSH
• sérový albumin MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lidský sérový albumin * MeSH
• makromolekulární látky MeSH
• nosiče léků MeSH
• sérový albumin MeSH

Multidrug resistance (MDR) represents one of the major concerns in cancer therapy as it may cause reduced efficacy of chemotherapeutic drugs due to the overexpression of ABC transporters, particularly P-glycoprotein (P-gp). This study explores the potential of novel amphiphilic diblock (DB) copolymers composed of poly[N-(2-hydroxypropyl)methacrylamide]-based copolymers (PHPMA) and poly(propylene oxide) (PPO) to overcome MDR mechanisms. The DB copolymers and their doxorubicin (Dox) conjugates significantly increased Dox accumulation in P-gp positive cells, markedly sensitizing them to Dox cytotoxic activity. The underlying mechanisms included depletion of intracellular ATP with subsequent inhibition of P-gp mediated drug efflux, an altered mitochondrial membrane potential, and increased ROS production. Moreover, the DB-Dox conjugates inhibited tumor growth in vivo more effectively compared to the corresponding PHPMA-based drug delivery system. Copolymers with additionally loaded PPO in the micelle core demonstrated superior efficacy in terms of P-gp inhibition, ATP depletion, and chemosensitizing effect in vitro, as well as antitumor activity in vivo. DB copolymers effectively depleted ATP levels both in vitro and in vivo using patient-derived xenograft (PDX) models, underscoring their capacity to enhance the effectiveness of standard chemotherapy and translational potential.

• Klíčová slova
• Diblock copolymers, Drug delivery system, HPMA copolymer, Intracellular ATP depletion, Multidrug resistance, P-glycoprotein inhibition, PPO, Sensitization to chemotherapy,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• chemorezistence účinky léků   MeSH
• doxorubicin * aplikace a dávkování   chemie   terapeutické užití   MeSH
• lidé MeSH
• methakryláty chemie   MeSH
• micely MeSH
• mnohočetná léková rezistence účinky léků   MeSH
• myši inbrední BALB C MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory farmakoterapie   metabolismus   patologie   MeSH
• nosiče léků * chemie   MeSH
• P-glykoprotein * metabolismus   MeSH
• polymery chemie   MeSH
• polypropyleny * chemie   MeSH
• propylenglykoly * chemie   aplikace a dávkování   MeSH
• protinádorová antibiotika * aplikace a dávkování   chemie   terapeutické užití   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• doxorubicin * MeSH
• methakryláty MeSH
• micely MeSH
• nosiče léků * MeSH
• P-glykoprotein * MeSH
• polymery MeSH
• polypropylene glycol MeSH Prohlížeč
• polypropyleny * MeSH
• propylenglykoly * MeSH
• protinádorová antibiotika * MeSH

New drug formulations are sought for poorly water-soluble substances because there is a risk of compromised bioavailability if such substances are administered orally. Such active pharmaceutical ingredients can be reformulated as solid dispersions with suitable water-soluble polymers. In this contribution, formulation of a novel and physically stable dispersion of Simvastatin in poly(2-hydroxypropyl) methacrylamide (pHPMA) is demonstrated. Due to the limited water sorption of pHPMA and a high Tg, the prepared dispersion is more suited for oral administration and storage compared with neat amorphous Simvastatin. Surprisingly, the rate of global reorientation and the internal motion of Simvastatin molecules were enhanced and exhibited dynamical heterogeneities when incorporated into the pHPMA matrix. As revealed by solid-state nuclear magnetic resonance combined with Raman spectroscopy exploiting the fluorescence phenomenon the mobility of the ester and lactone components increased considerably, whereas the naphthalene ring remained rigid. Furthermore, the solid dispersion was found to be nano-heterogeneous with nanometer-sized Simvastatin domains. The presence of these clusters had no impact on the dynamics of the rigid pHPMA chains. Thus, the diffusion of Simvastatin molecules through the glassy pHPMA walls and the subsequent transformation of the clusters into larger crystallites were prevented. No crystallization was detected for more than two years.

• Klíčová slova
• Fluorescence, Pharmaceuticals, Raman spectroscopy, Simvastatin, Solid dispersions, Solid-state NMR,
• MeSH
• adsorpce MeSH
• diferenciální skenovací kalorimetrie MeSH
• kyseliny polymethakrylové chemie   MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární struktura MeSH
• Ramanova spektroskopie MeSH
• simvastatin chemie   MeSH
• stabilita léku MeSH
• voda chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Duxon MeSH Prohlížeč
• kyseliny polymethakrylové MeSH
• simvastatin MeSH
• voda MeSH

INTRODUCTION: Fine needle aspiration biopsy (FNAB) is an easy method with an option of repetitive withdrawal of cell material. METHODS: First, mice were inoculated with mouse T-lymphoma, after 10 d the samples from tumor, lymph nodes and spleen gained by FNAB and excision were analyzed by flow cytometry. Tumor progression was compared to the control group simultaneously. Then, 10 d after tumor cell inoculation free doxorubicin (DOX) or different PHPMA DOX conjugates were injected. Cell material was analyzed to detect subpopulations of lymphocyte infiltrate, and levels of cytokines in correlation with progression or regression of the disease. RESULTS: FNAB has no influence on the tumor's growth or survival of experimental animals. After treatment with PHPMA conjugates there was a significant increase of T-lymphocyte subpopulations in tumor microenvironment compared to controls or free DOX, but only in mice with confirmed macroscopic regression of tumor within two weeks. Mice treated with conjugates showed significantly lower cancer infiltration of lymph nodes and spleen. CONCLUSION: FNAB provides a great benefit to in vivo monitoring of cell changes directly in the tumor after treatment. The number of infiltrating T-lymphocytes increases in correlation with consecutive tumor eradication after treatment with PHPMA. This proves that not only direct cytotoxic but also imunostimulating effect are necessary for successful treatment.

• MeSH
• doxorubicin aplikace a dávkování   chemie   terapeutické užití   MeSH
• experimentální nádory farmakoterapie   patologie   MeSH
• jehlová biopsie * MeSH
• kyseliny polymethakrylové chemie   MeSH
• metastázy nádorů * MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• proliferace buněk * MeSH
• protinádorové látky aplikace a dávkování   chemie   terapeutické užití   MeSH
• T-lymfocyty patologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• doxorubicin MeSH
• Duxon MeSH Prohlížeč
• kyseliny polymethakrylové MeSH
• protinádorové látky MeSH

The binding of plasma proteins to a drug carrier alters the circulation of nanoparticles (NPs) in the bloodstream, and, as a consequence, the anticancer efficiency of the entire nanoparticle drug delivery system. We investigate the possible interaction and the interaction mechanism of a polymeric drug delivery system based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers (pHPMA) with the most abundant proteins in human blood plasma-namely, human serum albumin (HSA), immunoglobulin G (IgG), fibrinogen (Fbg), and apolipoprotein (Apo) E4 and A1-using a combination of small-angle X-ray scattering (SAXS), analytical ultracentrifugation (AUC), and nuclear magnetic resonance (NMR). Through rigorous investigation, we present evidence of weak interactions between proteins and polymeric nanomedicine. Such interactions do not result in the formation of the protein corona and do not affect the efficiency of the drug delivery.

• Klíčová slova
• drug delivery, pHPMA, plasma proteins, polymeric nanoparticles, stealth effect,
• Publikační typ
• časopisecké články MeSH

• Publikační typ
• tisková chyba MeSH

• Publikační typ
• časopisecké články MeSH
• tisková chyba MeSH