Biomedical subjects

Pavla Kopecková

Publications and source records attributed to Pavla Kopecková.

At least 19 recordsLinked to original sources

PubMed2006

Biodistribution and pharmacokinetics of colon-specific HPMA copolymer--9-aminocamptothecin conjugate in mice.

A water soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-9-aminocamptothecin (9-AC) conjugate was designed for oral colon-specific drug delivery in the treatment of colon cancer. Comparative studies between the polymer conjugate and free drug have been performed to assess their biodistribution and pharmacokinetics in mice. After oral administration of equal doses of the polymer conjugate or free 9-AC, the drug concentrations in major organs at fixed time points were determined using an HPLC-fluorescence assay. Only 2+/-1% of 9-AC released from the polymer conjugate was detected in the small intestine (SI), and the mean peak concentration of free 9-AC was 45-fold higher than that from released drug. Colon-specific release of 9-AC produced high local concentrations. The mean peak concentration of released 9-AC in cecal contents, feces, cecal tissue, and colon tissue were, respectively, 3.2-fold, 3.5-fold, 2.2-fold and 1.6-fold higher than that using free 9-AC. In plasma, the high and sharp drug concentration profile from free drug was in contrast to the relatively low and flat pharmacokinetic profile obtained from drug released from the HPMA copolymer. There was no significant difference between released and free drug for the area under the concentration-time curve (AUC) and bioavailability values. As a consequence of the colon-specific release of unmodified 9-AC from the polymer conjugate, antitumor efficacy can be anticipated to be enhanced due to prolonged colon tumor exposure to higher and more localized drug concentrations.

Administration, Oral↗

PubMed2006

Hybrid hydrogels self-assembled from HPMA copolymers containing peptide grafts.

Graft copolymers were designed that self-assemble into hydrogels mediated by the interaction of coiled-coil peptide domains. A linear hydrophilic polymer of HPMA was chosen as the backbone, and coiled-coil forming peptides, covalently attached to the backbone, formed the grafts. Microrheology was used to evaluate the self-assembly of graft copolymers into hydrogels. The results revealed that the length and the number of coiled-coil grafts per chain had a significant influence on the gelation process. At least 4 heptads were needed to achieve the association of graft copolymers into hydrogels. CD spectra of the copolymer containing 5 heptad grafts further suggested that coiled-coil formation may contribute to the self-assembly. Gelation of graft copolymers containing CC4 peptides indicated that a threshold amount of grafts per macromolecule is needed to form a three-dimensional structure. These studies demonstrated a potential of the graft copolymers to create self-assembling hydrogels with desirable and controllable structures.

Amino Acid Sequence↗

PubMed2006

Water-soluble HPMA copolymer--prostaglandin E1 conjugates containing a cathepsin K sensitive spacer.

A novel bone targeting, N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer based, prostaglandin E1 (PGE1) delivery system was designed, synthesized and characterized. PGE1 was bound to the polymer backbone via a spacer, composed of a cathepsin K sensitive tetrapeptide (Gly-Gly-Pro-Nle) and a self-eliminating 4-aminobenzyl alcohol structure. The HPMA copolymer conjugates were prepared by photo-initiated free radical copolymerization of HPMA, PGE1-containing macromonomer, and optionally a comonomer containing a reactive p-nitrophenyl ester group. The latter group was used as attachment points for the D-aspartic acid octapeptide targeting moieties. Incubation of the PGE1-containing macromonomer and HPMA copolymer-PGE1 conjugates with cathepsin K resulted in release of unmodified PGE1. The rate of release depended on the composition of the conjugate. The higher the PGE1 content in the conjugate, the slower the PGE1 release. This appeared to be the result of association of hydrophobic side-chains in aqueous media, which rendered the formation of the enzyme substrate complex more difficult. The data seems to indicate that HPMA copolymer-PGE1 conjugates have a potential in the treatment of osteoporosis and other bone diseases.

Acrylamides↗

PubMed2005

Synthesis and characterization of novel aromatic azo bond-containing pH-sensitive and hydrolytically cleavable IPN hydrogels.

Novel interpenetrating network (IPN) hydrogels, composed of pH-sensitive, aromatic azo group containing network as one component (Network A), and a hydrolyzable network as the other (Network B), were prepared by a sequential process. The first network was formed by crosslinking of a reactive polymer precursor (copolymer of N,N-dimethylacrylamide, acrylic acid, N-tert.butylacrylamide, and N-methacryloylglycylglycine p-nitrophenyl ester) with an aromatic azo group containing diamine ((N,N'-epsilon-aminocaproyl)-4,4'-diaminoazobenzene). The second network was formed by radical crosslinking copolymerization of N-(2-hydroxypropyl)methacrylamide with N,O-dimethacryloylhydroxylamine. The composition of the hydrogels was manipulated to determine the influence of hydrogel composition on the equilibrium degree of swelling, modulus of elasticity in compression, and on the rate of degradation of Network B. Modeling of network structure was accomplished using the statistical branching theory. The major advantage of IPN hydrogels, when compared to traditional pH-sensitive networks, is the linear swelling profile following abrupt change of pH from 2 to 7.4. This indicates the suitability of IPN as carriers for oral drug delivery.

Absorption↗

PubMed2005

Bone-targeting macromolecular therapeutics.

Musculoskeletal diseases such as osteoporosis are recognized as major public health problems worldwide. Many novel therapeutic agents have been identified for the treatment of these diseases. However, the majority of them are not specific to hard tissue, resulting significant toxicity. Bone-targeting drug delivery systems based on water-soluble polymers can specifically direct candidate drugs to bone thereby reducing side effects due to non-specific tissue interactions. Incorporation of a targeting moiety, a drug release mechanism, drug selection and optimization of the polymer carrier are all essential elements in the development of bone-targeting macromolecular therapeutics. Successful clinical application of this approach can significantly contribute to the development of treatments for many musculoskeletal diseases.

Animals↗

PubMed2005

Biopolymer-based delivery systems for advanced imaging and skeletal tissue-specific therapeutics.

There is considerable advantage in developing tissue-specific delivery systems for therapeutic and diagnostic applications. Synthetic water-soluble polymeric delivery systems have been developed to allow selective delivery of therapeutic and imaging agents to musculoskeletal tissues. For mineralized tissues, bone-targeting agents such as aspartic acid octapeptide could concentrate the polymer conjugates to bone surfaces including resorption sites, which was demonstrated with routine bone histomorphometry. For bone-associated soft tissues, other targeting approaches based on pathophysiological properties unique to the local tissue environment, such as the leaky vasculature in arthritic joints, were utilized to achieve the selective deposition of the polymeric delivery systems to the desired sites. For this study, magnetic resonance imaging (MRI) was used to assess real-time pharmacokinetics and biodistribution of the MRI contrast agent conjugated polymer in major organs including skeletal tissues. The MRI data were then correlated with other standard imaging methods such as pQCT and DXA as well as routine histopathology and skeletal histomorphometry. Clearly, biopolymeric delivery systems may be used to improve the pharmacological and pharmacokinetic properties of different therapeutic agents for musculoskeletal diseases such as osteoporosis and arthritis. In addition, this or related technologies may also be useful to improve diagnosis and medical imaging with positron emission tomography, gamma scintigraphy, or other technologies.

Animals↗

PubMed2004

The arthrotropism of macromolecules in adjuvant-induced arthritis rat model: a preliminary study.

PURPOSE: To study the accumulation of macromolecules into the arthritic joints and the possible applications of such phenomenon. METHODS: The accumulation of plasma albumin in the joints of adjuvant-induced arthritis (AIA) rat model was first visualized with Evans blue injection. A N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer contrast agent was then synthesized and injected into the AIA rats to allow qualitative examination of biodistribution and pharmacokinetics of the injected macromolecule with magnetic resonance imaging (MRI). Vital organs and the diseased joints were isolated and examined histologically to correlate with the MRI findings. RESULTS: Deep blue color developed around the arthritic joints of the AIA rat a few hours after the injection of Evans blue. MR imaging of the AIA rats injected with polymer contrast agent demonstrated a gradual but very strong accumulation of the injected polymer in the arthritic joints, which lasted for 1-2 days. Observed differences in the concentration of the injected polymer in the joints correlated with disease severity as assessed histologically. CONCLUSIONS: Profound arthrotropism of macromolecules in the AIA rat model was demonstrated with various imaging tools. These observations should help in the conceptual and practical design of novel macromolecular delivery systems for the imaging and treatment of rheumatoid arthritis.

Animals↗

PubMed2003

Free and N-(2-hydroxypropyl)methacrylamide copolymer-bound geldanamycin derivative induce different stress responses in A2780 human ovarian carcinoma cells.

The effects of geldanamycin (GA), 17-(3-aminopropylamino)-17-demethoxygeldanamycin (AP-GA), and N-(2-hydroxypropyl)methacrylamide copolymer-AP-GA conjugate [P(AP-GA)] on A2780 human ovarian carcinoma cells at an equitoxic dose (2x IC(50)) were compared by the gene expression array analysis. All treatments resulted in similar gene expression profiles up to 12 h (e.g., down-regulation of CDK4 and up-regulation of APAF-1), although P(AP-GA)-treated cells showed delayed gene expression because of time-dependent internalization of the conjugate and intracellular drug release from P(AP-GA). However, AP-GA-treated cells showed elevated expression of HSP70 and HSP27 after 6 h compared with that observed by GA and P(AP-GA) treatments. Depletion of C-Raf, an HSP90 client protein, was observed in all treatments up to 12 h. Confocal microscopy using mesochlorin e(6) as a model drug revealed that drug release caused by the lysosomal cleavage of glycylphenylalanylleucylglycine oligopeptide spacer, used as GA derivative copolymer attachment/release point, was moderately fast. These results suggested that AP-GA treatment may activate stress-response pathways, whereas P(AP-GA) treatment may suppress them and trigger signaling pathways essential to cell growth arrest and death by inducing an HSP90-active factor. Although GA and P(AP-GA) treatments induced a time-dependent increase in HSP70 and HSP27 protein expression (detected by Western blotting analysis), AP-GA treatment resulted in more rapid and more intense expression of both proteins. Our results suggest that conjugation of AP-GA to N-(2-hydroxypropyl)methacrylamide copolymer may be able to modulate the cell stress responses induced by AP-GA because of differences in its internalization mechanism, subcellular localization, and intracellular concentration gradients.

Antibiotics, Antineoplastic↗

PubMed2003

Subcellular trafficking of HPMA copolymer-Tat conjugates in human ovarian carcinoma cells.

One of the main obstacles to efficient intracellular delivery of therapeutic macromolecules is the barrier posed by the plasma membrane. In this study, the cell penetrating peptide Tat was conjugated to a synthetic macromolecule based on N-(2-hydroxypropyl)methacrylamide (HPMA) and its subcellular distribution in human ovarian carcinoma cell lines was studied. The Tat peptide mediated uptake resulted in cytoplasmic and nuclear localization and was found to be energy independent. Time and concentration studies verified the rapidity and dependence of the transport process on these parameters. Enhanced uptake of a polymer bound anticancer drug doxorubicin was also demonstrated. These results were corroborated independently by subcellular fractionation.

Carcinoma↗

PubMed2003

Cytoplasmic delivery and nuclear targeting of synthetic macromolecules.

Delivery of macromolecular drugs (e.g. antisense oligonucleotides, polymer-drug conjugates, etc.) designed to work in specific sites inside cells is complicated as macromolecules typically have access to fewer biological compartments than small molecules. To better understand the fate of macromolecules in cells and begin to alter that fate, we investigated the internalization and subcellular fate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers and HPMA copolymer-drug conjugates in Hep G2 and A2780 cells. The subcellular fate of fluorescently labeled polymers was monitored by confocal microscopy and subcellular fractionation. Initially, the HPMA copolymers and HPMA copolymer-drug conjugates were internalized by endocytosis and remained in endosomes/lysosomes. At longer incubation times (>8 h), small amounts of the HPMA copolymers were observed to enter the cytoplasm and accumulate in the nucleus of the cells. Nuclear accumulation was confirmed after cytoplasmic microinjection. Oligonucleotides conjugated via lysosomally degradable spacers entered into the cytoplasm and nucleus of the cells faster than the polymers. The effect of the subcellular location was correlated to the toxicity of the photosensitizer, mesochlorin e(6) (Mce(6))-HPMA copolymer conjugates. The plasma membrane and late endosomes were more sensitive to damage by Mce(6). Targeting the polymer conjugates to the nucleus with the nuclear localization sequence (NLS) as well as conjugating the Mce(6) via a degradable spacer increased cell adhesion and uptake, promoted their entry into the cytoplasm and nucleus of the cells, and increased their toxicity. To further promote entry of the polymers into the cytoplasm and nucleus of the cells, the protein transduction domain, Tat peptide, was conjugated to the HPMA copolymers. This resulted in high binding to the cell membrane, but also facilitated rapid (<5 min) entry of the macromolecules into the cytoplasm and nucleus of cells. These results will prove valuable in the future design of macromolecular therapeutics.

Animals↗

PubMed2003

HPMA copolymer delivery of chemotherapy and photodynamic therapy in ovarian cancer.

Our studies document a unique and unexpected advantage of the combination of HPMA copolymer bound doxorubicin with mesochlorin e6/photodynamic therapy in the treatment of ovarian cancer. Each drug's activity is individually enhanced when compared with free (low molecular weight) drugs, furthermore, in combination these HPMA copolymer bound agents act synergistically to create an unexpected biological effect. Figure 8 depicts the known activities of each agent which may play synergistic roles. HPMA copolymer-doxorubicin has been widely evaluated in preclinical and clinical studies. It demonstrates marked advantages over free doxorubicin: control of biodistribution and accumulation via molecular weight restrictions, biodegradability, minimal immunogenicity, subcellular localization, anticancer activity, enhanced permeability and retention, increased apoptosis, lipid peroxidation, DNA damage, and reduced nonspecific toxicity. Recent clinical trials in the UK provide "proof of principle" of the "enhanced permeability and retention effect" for solid tumors and the unique advantages of this novel drug delivery system for the treatment of ovarian cancer. With regards to photodynamic therapy using the photosensitizer mesochlorin e6, the preclinical evaluations thus far document: control of biodistribution and accumulation via molecular weight restrictions, biodegradability, subcellular localization, anticancer activity, enhanced permeability and retention, and reduced nonspecific toxicity. Ongoing microarray studies document unique cellular pathways and new pharmaceutical properties which are initiated by the HPMA copolymer delivery delivery of these agents, and predict an exciting future for this novel drug delivery system.

Antibiotics, Antineoplastic↗

PubMed2003

Correlation of subcellular compartmentalization of HPMA copolymer-Mce6 conjugates with chemotherapeutic activity in human ovarian carcinoma cells.

PURPOSE: Intracellular targets sensitive to oxidized damage generated by photodynamic therapy (PDT) utilizing N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-mesochlorin e6 monoethylenediamine (Mce6) conjugates was explored to aid in the design of second generation PDT delivery systems. METHODS: Low temperature, metabolic inhibitor, and nuclear localization sequences (NLS(FITC)) were used to achieve desired subcellular localization that was evaluated by confocal analysis and subcellular fractionation. Mce6 was bound to HPMA copolymer conjugates via non-degradable dipeptide linkers (P-GG-Mce6, P-NLS(FITC)-GG-Mce6) or lysosomally degradable tetrapeptide spacers (P-GFLG-Mce6, P-NLS(FITC)-GFLG-Mce6). Chemotherapeutic efficacy was assessed by the concentration that inhibited growth by 50% (IC50), cell associated drug concentration (CAD) and confocal microscopy. RESULTS: P-GFLG-Mce6 possessed enhanced chemotherapeutic activ ity compared to P-GG-Mce6 indicating enzymatically released Mce6 was more active than copolymer-bound Mce6. Lysosomes appeared less sensitive to photodamage as observed by a higher IC50. Nuclear-directed HPMA copolymer-Mce6 conjugates (P-NLS(FITC)-GG-Mce6, P-NLS(FITC)-GFLG-Mce6) possessed enhanced chemotherapeutic activity. However, control cationic HPMA copolymer-Mce6 conjugates containing a scrambled NLS (P-scNLS(FITC)-GG-Mce6) or amino groups (P-NH2-GG-Mce6) also displayed increased chemotherapeutic activity. CONCLUSIONS: Nuclear delivery was observed for P-NLS(FITC)-GG-Mce6 and P-NLS(FITC)-GFLG-Mce6 indicating NLS was a feasible approach for nuclear delivery. Due to the cationic nature of NLS, increased membrane binding of PDT systems incorporating cationic nuclear targeting moieties must be addressed.

Cell Line, Tumor↗

PubMed2003

Mechanisms of cytotoxicity in human ovarian carcinoma cells exposed to free Mce6 or HPMA copolymer-Mce6 conjugates.

It is essential to understand cellular responses on photodynamic therapy (PDT) to design delivery systems that maximize cytotoxic effects coupled with minimal induction of side effects or protective mechanisms (or both). Here, we investigated mechanisms of toxicity in human ovarian carcinoma A2780 cells treated with structurally diverse N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (P)-mesochlorin e6 monoethylenediamine (Mce6) conjugates that possessed differential subcellular accumulation or covalent attachments of photosensitizers (or both). Apoptosis and necrosis were observed after photoactivation, with increased apoptotic responses observed in cells exposed to conjugates possessing Mce6 linkage via a lysosomally degradable tetrapeptide spacer (HPMA copolymer-Mce6 conjugates containing Mce6 bound via glycylphenylalanylleucylglycine [GFLG] linker [P-GFLG-Mce6], HPMA copolymer-Mce6 conjugates containing Mce6 bound via a GFLG spacer and containing nuclear localization sequence, PKKKRKV132K(FITC)C [NLS(fluorescein-5-isothiocyanate [FITC])] bound via a thioether linkage [P-NLS(FITC)-GFLG-Mce6]). Furthermore, the induction of necrosis was more pronounced in cells exposed to conjugates containing both a nuclear localization sequence (NLS) and Mce6 bound by a degradable linker (P-NLS(FITC)-GFLG-Mce6). Caspase-independent mechanisms of cell death were identified in cells treated with nuclear-targeted conjugates possessing Mce6 attached using a nondegradable tether (HPMA copolymer-Mce6 conjugates containing Mce6 bound via a GG spacer and containing NLS(FITC) bound via a thioether linkage [P-NLS(FITC)-GG-Mce6]), whereas low levels of apoptosis and necrosis were detected in cells exposed to photoactivated nontargeted HPMA copolymer-Mce6 conjugates containing Mce6 coupled through a nondegradable spacer (HPMA copolymer-Mce6 conjugates containing Mce6 bound via GG linker [P-GG-Mce6]). Variations in gene expression were observed in cells on PDT. Specifically, HSP70 expression was solely detected in cells treated with P-GFLG-Mce6, whereas the loss of detection of several genes were observed in cells treated with P-NLS(FITC)-GFLG-Mce6. Variations in cellular responses on PDT using different HPMA copolymer-Mce6 conjugates will prove useful in the design of optimal HPMA copolymer PDT delivery systems.

Apoptosis↗

PubMed2002

Inhibition of cathepsin K with lysosomotropic macromolecular inhibitors.

Cathepsin K is the major enzyme responsible for the degradation of the protein matrix of bone and probably for the destruction of articular cartilage in rheumatoid arthritis joints. These processes occur mainly in the resorption lacuna and within the lysosomal compartment. Here, we have designed, synthesized, and evaluated new lysosomotropic (water-soluble) polymer-cathepsin K inhibitor conjugates. In particular, we characterized the relationship between conjugate structures and their activity to inhibit cathepsins K, B, L, and papain. A potent selective cathepsin K inhibitor, 1,5-bis(N-benzyloxycarbonylleucyl)carbohydrazide, was modified to 1-(N-benzyloxycarbonylleucyl)-5-(phenylalanylleucyl)carbohydrazide (I) to facilitate polymer conjugation. It was conjugated to the polymer chain termini of two water-soluble polymers [alpha-methoxy poly(ethylene glycol), abbreviated as mPEG-I; semitelechelic poly[N-(2-hydroxypropyl)methacrylamide], abbreviated as ST-PHPMA-I]. The conjugation of inhibitor I to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer side chains was accomplished via either a Gly-Gly spacer (PHPMA-GG-I) or with no spacer between I and the copolymer backbone (PHPMA-I). Kinetic analysis revealed that free inhibitor I possessed an apparent second-order rate constant against cathepsin K (k(obs)/[I] = 1.3 x 10(6) M(-1) s(-1)) similar to that of unmodified 1,5-bis(Cbz-Leu) carbohydrazide, while I conjugated to the chain termini of mPEG and ST-PHPMA-COOH had slightly lower values (about 5 x 10(5) M(-1) s(-1)). The k(obs)/[I] values for I attached to the side chains of HPMA copolymers (PHPMA-GG-I and PHPMA-I) were about 3 x 10(4) M(-1) s(-1). When tested against cathepsin L, inhibitor I and all its polymer conjugates produced k(obs)/[I] values 1-2 orders of magnitude less than those determined for cathepsin K, while for cathepsin B and papain, the values were 2-4 orders of magnitude lower. The ability of mPEG-I and ST-PHPMA-I to inhibit cathepsin K activity in synovial fibroblasts was also evaluated. Both polymer-bound inhibitors were internalized by endocytosis and were ultimately trafficked to the lysosomal compartment. ST-PHPMA-I was internalized faster than mPEG-I. The inhibitory activity in the synovial fibroblast assay correlated with the rate of internalization.

Cathepsin K↗

PubMed2002

Design of novel bioconjugates for targeted drug delivery.

This paper summarizes recent work on the design and development of targeted polymeric bioconjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. Polymerizable antibody Fab' fragment (MA-Fab') has been developed and used in the preparation of targeted HPMA copolymer-mesochlorin e6 conjugates for the treatment of human ovarian carcinomas. The reactivity of the MA-Fab' in copolymerization with HPMA depended on the length of the spacer between the monomer double bond and the antibody Fab' fragment. The biological activity of the antibody Fab' fragment was maintained after incorporation into the HPMA copolymer. Novel aromatic azo spacers were designed and incorporated into HPMA copolymer-drug (cyclosporin A, 9-aminocamptothecin) conjugates for the colon-specific drug delivery and for the treatment of colon diseases. The colon-specific drug release from the conjugates was controlled by the structures of both drug and spacers. Lectins, wheat germ agglutinin (WGA) and peanut agglutinin (PNA), were conjugated to the colon-specific polymer drug conjugates to enhance specific adhesion onto colon tissues.

Colonic Diseases↗

PubMed2002

Influence of the structure of drug moieties on the in vitro efficacy of HPMA copolymer-geldanamycin derivative conjugates.

PURPOSE: To optimize the structure of geldanamycin (GDM) derivative moieties attached to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers via an enzymatically degradable spacer. METHODS: HPMA copolymers containing different AR-GDM (AR = 3-aminopropyl (AP), 6-aminohexyl (AH), and 3-amino-2-hydroxypropyl AP(OH)) were synthesized and characterized. Their cytotoxicity towards the A2780 human ovarian carcinoma cells was evaluated. RESULTS: The cytotoxic efficacy of HPMA copolymer-AR-GDM conjugates depended on the structure of AR-GDM. Particularly, HPMA copolymer-bound AH-GDM, which possessed the longest substituent at the 17-position, demonstrated the highest efficacy among the polymer-bound GDM derivatives; however the activity of free AH-GDM was lower than that of the other free AR-GDMs. The relative increase of the activity of macromolecular AH-GDM when compared to AP-GDM or AP(OH)-GDM correlated with the enhanced recognition of AH-GDM terminated oligopeptide side-chains by the active site of the lysosomal enzyme, cathepsin B. Drug stability and further stabilization upon binding to HPMA copolymer also contributed to the observed phenomena. CONCLUSIONS: AH-GDM was found to be a suitable GDM derivative for the design of a drug delivery system based on HPMA copolymers and enzymatically-degradable spacers.

Acrylamides↗

PubMed2002

The role of galactose, lactose, and galactose valency in the biorecognition of N-(2-hydroxypropyl)methacrylamide copolymers by human colon adenocarcinoma cells.

PURPOSE: To examine the beta-galactoside and beta-lactoside binding capacity of three human colon-adenocarcinoma cell lines and their sugar specificity, using N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates of galactosamine, lactose, and triantennary galactose. METHODS: Three types of HPMA copolymers containing the saccharide epitopes galactosamine (P-Gal), lactose (P-Lac), or triantennary galactose (P-TriGal) were synthesized. The relationship between the content of the saccharide moieties, the valency of the galactose residues, and their biorecognition by the cell lines (Colo-205, SW-480, and SW-620) was investigated using flow cytometry and confocal fluorescence microscopy analysis. RESULTS: The binding of the glycoconjugates to the human colonadenocarcinoma cell lines was dependent on the type and the number of bound sugar residues per macromolecule. The higher the sugar contents in the HPMA copolymers, the higher the extent of binding. Although introduction of galactoside residues into the HPMA copolymer resulted in a significant increase in the binding of the copolymers to the cells, low biorecognition of the lactoside-containing HPMA copolymers by all cell lines used was observed. The trivalent galactoside-containing HPMA copolymers did not yield a notable glycoside cluster effect for the beta-galactoside-binding lectin expressed on human colon-adenocarcinoma cells. Among the various cell line little differences in the extent of binding of the glycopolymers to the cells were observed. The data on the internalization of HPMA copolymer conjugates obtained by confocal fluorescence microscopy correlated well with the flow cytometry analysis of their biorecognition by target cells. CONCLUSIONS: The lectin-mediated endocytosis of the HPMA glycoconjugates in human colon cancer cell lines suggests their potential use as targeting tools of cytotoxic drugs to colon adenocarcinoma.

Adenocarcinoma↗

PubMedDate not supplied

Antisense oligonucleotides delivered to the lysosome escape and actively inhibit the hepatitis B virus.

The subcellular fate and activity in inhibiting the hepatitis B virus of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-phosphorothioate oligonucleotides were studied. Their internalization and subcellular fate were monitored with confocal microscopy. A fraction of the internalized free oligonucleotides escaped into the cytoplasm and nucleus of Hep G2 cells but were not active antiviral agents. Covalently attaching the oligonucleotides to the HPMA copolymers via nondegradable dipeptide GG spacers resulted in sequestering the oligonucleotides in vesicles after internalization. Conjugation of the oligonucleotides to an HPMA copolymer via a lysosomally cleavable tetrapeptide GFLG spacer resulted in release of the oligonucleotide in the lysosome and subsequent translocation into the cytoplasm and nucleus of the cells. The HPMA copolymer-oligonucleotide conjugate possessed antiviral activity, indicating that phosphorothioate oligonucleotides released from the carrier in the lysosome were able to escape into the cytoplasm and nucleus and remain active. The Hep G2 cells appeared to actively internalize the phosphorothioate oligonucleotides as oligonucleotide-HPMA copolymer conjugates were internalized to a greater extent than unconjugated polymers.

Antiviral Agents↗