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1.
Children (Basel) ; 10(4)2023 Apr 19.
Article in English | MEDLINE | ID: mdl-37189991

ABSTRACT

Restricted and repetitive behaviors and interests (RRBI) are a significant component in diagnosing autism spectrum disorder (ASD). They often pose the main challenge in day-to-day functions for children with ASD and their families. Research addressing family accommodation behaviors (FAB) in the ASD population is scarce, and associations with the characteristics of the children's behaviors are unclear. This sequential mixed-methods study assessed the correlation between RRBI and FAB within the ASD group to deepen the understanding of parents' subjective experiences regarding their children's RRBI. It included a quantitative phase with a follow-up qualitative study. A total of 29 parents of children with autism (5-13 yr) completed the study questionnaires; a total of 15 also were interviewed regarding their children's RRBI and related FAB. We used the Repetitive Behavior Scale-Revised (RBS-R) to assess RRBI, and the Family Accommodation Scale (FAS-RRB) to assess FAS. In-depth interviews from phenomenological methodology were used in the qualitative phase. We found significant positive correlations between the RRBI and FAB overall and their subscores. Qualitative research supports these findings, adding descriptive examples of the accommodations families make to address the RRBI-related challenges. The results indicate relations between RRBI and FAB and the importance of practically addressing children with autism's RRBI and their parents' experiences. Both affect and are affected by the children's behaviors.

2.
Front Med Technol ; 4: 846065, 2022.
Article in English | MEDLINE | ID: mdl-35463298

ABSTRACT

Endothelial cell adhesion molecules have long been proposed as promising targets in many pathologies. Despite promising preclinical data, several efforts to develop small molecule inhibitors or monoclonal antibodies (mAbs) against cell adhesion molecules (CAMs) ended in clinical-stage failure. In parallel, many well-validated approaches for targeting CAMs with nanomedicine (NM) were reported over the years. A wide range of potential applications has been demonstrated in various preclinical studies, from drug delivery to the tumor vasculature, imaging of the inflamed endothelium, or blocking immune cells infiltration. However, no NM drug candidate emerged further into clinical development. In this review, we will summarize the most advanced examples of CAM-targeted NMs and juxtapose them with known traditional drugs against CAMs, in an attempt to identify important translational hurdles. Most importantly, we will summarize the proposed strategies to enhance endothelial CAM targeting by NMs, in an attempt to offer a catalog of tools for further development.

3.
J Control Release ; 330: 1191-1207, 2021 02 10.
Article in English | MEDLINE | ID: mdl-33207257

ABSTRACT

The vast majority of nanomedicines (NM) investigated today consists of a macromolecular carrier and a drug payload (conjugated or encapsulated), with a purpose of preferential delivery of the drug to the desired site of action, either through passive accumulation, or by active targeting via ligand-receptor interaction. Several drug delivery systems (DDS) have already been approved for clinical use. However, recent reports are corroborating the notion that NM do not necessarily need to include a drug payload, but can exert biological effects through specific binding/blocking of important target proteins at the site of action. The seminal work of Kopecek et al. on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing biorecognition motifs (peptides or oligonucleotides) for crosslinking cell surface non-internalizing receptors of malignant cells and inducing their apoptosis, without containing any low molecular weight drug, led to the definition of a special group of NM, termed Drug-Free Macromolecular Therapeutics (DFMT). Systems utilizing this approach are typically designed to employ pendant targeting-ligands on the same macromolecule to facilitate multivalent interactions with receptors. The lack of conventional small molecule drugs reduces toxicity and adverse effects at off-target sites. In this review, we describe different types of DFMT that possess biological activity without attached low molecular weight drugs. We classified the relevant research into several groups by their mechanisms of action, and compare the advantages and disadvantages of these different approaches. We show that identification of target sites, specificity of attached targeting ligands, binding affinity and the synthesis of carriers of defined size and ligand spacing are crucial aspects of DFMT development. We further discuss how knowledge in the field of NM accumulated in the past few decades can help in the design of a successful DFMT to speed up the translation into clinical practice.


Subject(s)
Pharmaceutical Preparations , Drug Carriers , Drug Delivery Systems , Ligands , Macromolecular Substances , Nanomedicine , Polymers
4.
J Control Release ; 319: 475-486, 2020 03 10.
Article in English | MEDLINE | ID: mdl-31838202

ABSTRACT

Inflammation with neutrophils infiltration is a prominent feature of alcohol-related liver disease (ARLD) and contributes to the severity of liver injury. Although an array of potential treatments has been studied, the standard treatment regimen is controversial and can induce severe side effects and infection-related complications. E-selectin, a cytokine inducible cell adhesion molecule, mediates the initial interaction of leucocytes with endothelial cells, and facilitates their further adhesion and extravasation into inflamed tissues. Given the important role of E-selectin in leukocytes trafficking, we hypothesized that a synthetic polymer presenting multiple copies of E-selectin binding peptide in a polyvalent manner (P-Esbp) may block the "roads" that facilitate neutrophil infiltration, inhibit the recruitment of neutrophils to the inflamed sites and reduce the extent of liver injury. We now demonstrate in vitro that P-Esbp reduced the recruitment of neutrophils (collected from blood of donors) on activated human umbilical vein endothelial cells (HUVEC) under flow conditions. Pre-treatment of mice with P-Esbp prior to alcohol binge attenuated alcohol-induced serum transaminase (ALT, AST) elevation, reduced pro-inflammatory cytokines (TNFα and IL-1ẞ) and chemokines (MIP-2/CXCL2 and MCP-1/CCL2) in National Institute on Alcohol Abuse and Alcoholism (NIAAA) model. Also, the up-regulation of neutrophil marker Ly6G and the number of MPO positive cells in the injured tissue was significantly reduced by the treatment, indicating diminished neutrophil infiltration. Moreover, as a result of P-Esbp treatment, E-selectin expression in the liver (mRNA and protein level) was downregulated, suggesting a potential to decrease ongoing local inflammatory response. Overall, our findings highlight the anti-inflammatory properties of the "drug-free" P-Esbp and its therapeutic potential to attenuate an excessive inflammation where infiltrating neutrophils can damage tissues and organs.


Subject(s)
E-Selectin , Neutrophils , Animals , Cell Adhesion , Endothelial Cells , Liver , Mice , Polymers
5.
Biomacromolecules ; 20(9): 3547-3556, 2019 09 09.
Article in English | MEDLINE | ID: mdl-31381303

ABSTRACT

Detection and removal of preneoplastic tumors is crucial for successful colorectal cancer (CRC) therapy. Here we describe the design of a Cathepsin B (CB)-activated polymeric probe, P-(GGFLGK-IR783), for imaging CRC tumors established by intrarectal or subcutaneous (s.c.) implantation of human colon cancer cells (SW-480 and HT-29) in mice. Multiple copies of the near-infrared fluorescent (NIRF) dye IR783 were attached to a single HPMA copolymer backbone via a CB-cleavable linker (GFLG), and the influence of the dye loading on the fluorescence quenching and activation by CB was assessed in vitro, ex vivo, and in vivo. The optimal dose and dosing regimen of P-(GGFLGK-IR783) for colonic tumor detection was determined. Increasing the IR783 loading in the copolymer from 2.5 to 20 mol % resulted in quenching of the fluorescence signal that was activated in vitro by the action of CB from different origins. Following intravenous administration, P-(GGFLGK-IR783)7.5% preferentially accumulated in intrarectal and s.c. implanted tumors, allowing tumor visualization after 4 h and even 48 h postadministration. Activation of P-(GGFLGK-IR783)7.5% by CB was clearly detected in s.c. implanted tumors, revealing about a 4-fold increase in the fluorescence signal in tumors vs healthy colon tissue. The probe containing the CB-cleavable linker produced higher fluorescence signal intensity in tumors, relative to the noncleavable probe. These results indicate that P-(GGFLGK-IR783)7.5% may aid in detecting CRC tumors and can help to guide selective removal of polyps during colonoscopic procedures.


Subject(s)
Cathepsin B/pharmacology , Colorectal Neoplasms/diagnostic imaging , Fluorescent Dyes/pharmacology , Molecular Imaging , Animals , Cathepsin B/chemistry , Colorectal Neoplasms/pathology , Fluorescent Dyes/chemistry , HT29 Cells , Heterografts , Humans , Mice , Polymers/chemistry , Polymers/pharmacology , Spectroscopy, Near-Infrared
6.
J Control Release ; 288: 136-147, 2018 10 28.
Article in English | MEDLINE | ID: mdl-30165141

ABSTRACT

Endothelial activation with up-regulation of E-selectin adhesion molecules mediates leukocyte rolling along the vascular wall and controls inflammation in many diseases including atherosclerosis and heart failure. Therefore, we aimed to test the hypothesis that inhibition of E-selectin-mediated interactions by a new E-selectin-targeted copolymer could inhibit the progression of atherosclerosis. To target E-selectin on activated endothelium, we developed a new N-(2-hydroxypropyl)methacrylamide (HPMA)-based E-selectin binding copolymer with or without dexamethasone (Dex) (designated P-(Esbp)-Dex and P-Esbp, respectively). To determine the effect of P-(Esbp)-Dex and P-Esbp on atherosclerosis, we allocated ApoE (-/-) mice on a high fat diet, to weekly intra-peritoneal injections of either 1) P-Esbp; 2) P-(Esbp)-Dex; 3) free Dex (1 mg/kg) or 4) saline, for four weeks. Aortic atherosclerosis and left ventricular (LV) remodeling and function were assessed by serial ultrasound studies and histology. Monocytes and macrophages were characterized by flow cytometry. After four weeks of treatment, P-Esbp effectively targeted aortic atherosclerotic lesions. Both P-Esbp and P-(Esbp)-Dex reduced wall thickening of the ascending aortas. However, only the drug-free copolymer (P-Esbp) significantly decreased the areas of necrotic core in the plaques and switched spleen macrophages toward an anti-inflammatory (M2) phenotype. Furthermore, P-Esbp attenuated adverse LV remodeling and dysfunction in ApoE (-/-) mice. In summary, P-Esbp copolymer targets activated endothelial cells, regresses and stabilizes atherosclerotic plaques, and prevents adverse LV remodeling and dysfunction in ApoE (-/-) mice. Our results suggest a new, drug-free macromolecular therapy to treat vascular inflammation.


Subject(s)
Anti-Inflammatory Agents/administration & dosage , Atherosclerosis/drug therapy , E-Selectin/antagonists & inhibitors , Methacrylates/administration & dosage , Ventricular Dysfunction, Left/drug therapy , Ventricular Remodeling/drug effects , Animals , Aorta/drug effects , Aorta/metabolism , Atherosclerosis/metabolism , Atherosclerosis/pathology , Atherosclerosis/physiopathology , Dexamethasone/administration & dosage , E-Selectin/metabolism , Macrophages/drug effects , Male , Mice, Inbred C57BL , Mice, Knockout, ApoE , Monocytes/drug effects , Ventricular Dysfunction, Left/metabolism , Ventricular Dysfunction, Left/pathology , Ventricular Dysfunction, Left/physiopathology
7.
Mol Pharm ; 15(9): 3690-3699, 2018 09 04.
Article in English | MEDLINE | ID: mdl-29957956

ABSTRACT

One of the greatest challenges in cancer therapy is to control metastatic spread, seeding, and growth of tumors in distant organs. Recently, we reported on the design of a novel "drug-free" therapeutic copolymer bearing the antimigratory A5G27 peptide, designated P-(A5G27)-FITC, that shows excellent specificity to cancer cells overexpressing CD44v3 and CD44v6 and inhibits cancer cell migration and invasion. We demonstrated that P-(A5G27)-FITC accumulated preferentially in subcutaneous (sc) implanted 4T1 tumors following parenteral administration. Moreover, we showed that pretreatment of mice with P-(A5G27)-FITC prior to 4T1 cell inoculation inhibited colonization of circulating 4T1 cells in the lungs. In this study, we designed a new polymer-peptide-drug conjugate to inhibit vigorously growing primary tumors and control invasive behavior of cancer cells. To this end, the antimitotic drug (paclitaxel, PTX) was conjugated to P-(A5G27)-FITC. The targeted polymer-drug conjugate (P-(A5G27)-PTX) was significantly more toxic toward CD44-overexpressing cancer cells than the nontargeted copolymer. In vivo, a single iv injection of P-(A5G27)-PTX prolonged the survival of C57BL/6 mice with established B16-F10 lung metastases. When injected intraperitoneally into BALB/c mice implanted sc with 4T1 tumors, P-(A5G27)-PTX significantly decreased the rate of primary tumor growth, increased the median survival of mice, and reduced the number of 4T1 metastases in the lungs when compared to nontargeted copolymer. Most interestingly, the CD44-targeted "drug-free" copolymer P-(A5G27) (without PTX) significantly inhibited the rate of tumor growth and further prolonged the median survival time of mice to the same extent as the PTX-containing formulations (P-(A5G27)-PTX or free PTX). Overall, this study highlights the therapeutic potential of the HPMA copolymer-A5G27 conjugates ("drug-free" and PTX-bearing copolymers) to control the metastatic spread of cancer.


Subject(s)
Drug Delivery Systems/methods , Paclitaxel/chemistry , Polymers/chemistry , Animals , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/physiology , Cell Survival/drug effects , Female , Hyaluronan Receptors/metabolism , Mammary Neoplasms, Animal/drug therapy , Mammary Neoplasms, Animal/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Paclitaxel/therapeutic use
8.
Adv Drug Deliv Rev ; 119: 120-142, 2017 09 15.
Article in English | MEDLINE | ID: mdl-28506743

ABSTRACT

Since their initial discovery more than 30years ago, tumor-homing peptides have become an increasingly useful tool for targeted delivery of therapeutic and diagnostic agents into tumors. Today, it is well accepted that cells at the tumor microenvironment (TME) contribute in many ways to cancer development and progression. Tumor-homing peptide-decorated nanomedicines can interact specifically with surface receptors expressed on cells in the TME, improve cellular uptake of nanomedicines by target cells, and impair tumor growth and progression. Moreover, peptide ligand-modified nanomedicines can potentially accumulate in the target tissue at higher concentrations than would small conjugates, thus increasing overall target tissue exposure to the therapeutic agent, enhance therapeutic efficacy and reduce side effects. This review describes the most studied peptide ligands aimed at targeting cells in the TME, discusses major obstacles and principles in the design of ligands for drug targeting and provides an overview of homing peptides in ligand-targeted nanomedicines that are currently in development for cancer therapy and diagnosis.


Subject(s)
Nanoparticles/administration & dosage , Peptides/administration & dosage , Tumor Microenvironment/drug effects , Animals , Drug Delivery Systems/methods , Humans , Ligands , Nanomedicine/methods
9.
J Control Release ; 257: 10-20, 2017 07 10.
Article in English | MEDLINE | ID: mdl-28093296

ABSTRACT

The prevention of cancer cell dissemination and secondary tumor formation are major goals of cancer therapy. Here, we report on the development of a new CD44-targeted copolymer carrying multiple copies of the A5G27 peptide, known for its ability to bind specifically to CD44v3 and CD44v6 on cancer cells and inhibit tumor cell migration, invasion, and angiogenesis. We hypothesized that conjugation of A5G27 to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer would enhance tumor tissue accumulation, promote selective binding to cancer cells, with concomitant increased inhibition of cancer cell invasiveness and migration. Fluorescein-5-isothiocyanate or the near-infrared fluorophore IR783 were attached to the copolymer backbone through a non-cleavable linkage to assess in vitro binding to cancer cells and biodistribution of the polymer in 4T1 murine mammary adenocarcinoma-bearing mice, respectively. The anti-migratory activity was evaluated both in vitro and in vivo. The binding of the targeted copolymer to cancer cells correlated well with the level of CD44 expression, with the polymer being internalized more efficiently by cancer cells. Pre-treatment of mice with polymer-bound A5G27 significantly inhibited lung colonization of migrating 4T1 cells in vivo, with the targeted copolymer accumulating preferentially in subcutaneous 4T1 tumors, when compared to a non-targeted system. As such, the HPMA copolymer-A5G27 conjugate is a promising candidate for inhibiting cancer cell migration and can also be used as a drug or imaging probe carrier for detection and treatment of cancer.


Subject(s)
Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Hyaluronan Receptors/antagonists & inhibitors , Neoplasm Invasiveness/prevention & control , Peptides/therapeutic use , Prostatic Neoplasms/drug therapy , Acrylamides/chemistry , Acrylamides/pharmacokinetics , Acrylamides/therapeutic use , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Breast Neoplasms/pathology , Cell Movement/drug effects , Drug Delivery Systems , Female , Humans , Male , Mice , Mice, Inbred BALB C , Neoplasm Invasiveness/pathology , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/pathology , Peptides/chemistry , Peptides/pharmacokinetics , Prostatic Neoplasms/pathology , Tissue Distribution
10.
Eur J Pharm Biopharm ; 109: 103-112, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27702685

ABSTRACT

The key for successful gene silencing is to design a safe and efficient siRNA delivery system for the transfer of therapeutic nucleic acids into the target cells. Here, we describe the design of hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer displaying multiple copies of octaarginine (R8) and its use in promoting the effective delivery of small interfering RNA (siRNA) molecules intracellularly. Fluorescein-5-isothiocyanate (FITC)-labeled HPMA copolymer-bound R8 (P-R8-FITC) was synthesized with increasing R8 molar ratios (4-9.5mol-%) to define the optimal R8 content that allowed the polymer to serve both as a siRNA-binding domain and as an intracellular transduction moiety mediating improved cellular delivery. A subunit of the influenza virus hemagglutinin (HA2), known for its ability to disrupt endosomal membranes, was further conjugated to P-R8-FITC copolymer to promote endosomal escape. Of the different P-(R8)-FITC conjugates considered, only that polymer containing the highest mol-% of R8 (P-(R8)9.5-FITC) was able to encapsulate siRNA molecules into nano-sized polyion complexes (PICs) presenting positive surface charge, low in vitro cytotoxicity, and high serum stability. P-(R8)9.5-FITC/cy5-siRNA complexes can efficiently deliver siRNA molecules into cells, while naked siRNA or siRNA encapsulated within polymers with lower R8mol-% were unable to transfect the same cells. Conjugation of HA2 fusogenic peptide to P-(R8)-FITC significantly decreased the oncogenic RAC1 mRNA levels in cancer cells. This indicates that P-(R8)-(HA2)-FITC can deliver siRNA into target cells, and that the siRNA can reach the perinuclear region where it interacts with the RNA-induced silencing complex.


Subject(s)
Acrylamides/chemistry , Gene Silencing , Neoplasms/genetics , Oligopeptides/chemistry , Polymers/chemistry , RNA, Small Interfering/genetics , A549 Cells , Cell Line, Tumor/drug effects , Cytoplasm/metabolism , Endosomes/metabolism , Female , Fluorescein-5-isothiocyanate/chemistry , Gene Expression Regulation, Neoplastic , Gene Transfer Techniques , Humans , Microscopy, Confocal , Ovarian Neoplasms/genetics , Peptides/chemistry , RNA/chemistry , RNA Interference , Real-Time Polymerase Chain Reaction , Surface Properties
11.
J Control Release ; 229: 192-199, 2016 05 10.
Article in English | MEDLINE | ID: mdl-27001892

ABSTRACT

Polymer-drug conjugates that can actively target the tumor vasculature have emerged as an attractive technology for improving the therapeutic efficacy of cytotoxic drugs. We have recently provided, for the first time, in vivo evidence showing the significant advantage of the E-selectin-targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin conjugate, P-(Esbp)-DOX, in inhibiting primary tumor growth and preventing the formation and development of cancer metastases. Here, we describe the design of a vascular endothelial growth factor receptor (VEGFR)-1-targeted HPMA copolymer-DOX conjugate (P-(F56)-DOX) that can actively and simultaneously target different cell types in the tumor microenvironment, such as endothelial cells (ECs), bone marrow-derived cells and many human cancer cells of diverse tumor origin. The VEGFR-1-targeted copolymer was tested for its binding, internalization and in vitro cytotoxicity in ECs (bEnd.3 and cEND cells) and cancer cells (B16-F10, 3LL and HT29). The in vivo anti-cancer activity of P-(F56)-DOX was then tested in two tumor-bearing mice (TBM) models (i.e., primary Lewis lung carcinoma (3LL) tumors and B16-F10 melanoma pulmonary metastases), relative to that of the E-selectin-targeted system (P-(Esbp)-DOX) that solely targets ECs. Our results indicate that the binding and internalization profiles of the VEGFR-1-targeted copolymer were superior towards ECs as compared to cancer cells and correlated well to the level of VEGFR-1 expression in cells. Accordingly, the VEGFR-1-targeted copolymer (P-(F56)-DOX) was more toxic towards bEnd.3 cells than to cancer cells, and exhibited significantly higher cytotoxicity than did the non-targeted control copolymer. P-(F56)-DOX inhibited 3LL tumor growth and significantly prolonged the survival of mice with B16-F10 pulmonary metastases. When compared to a system that actively targets only tumor vascular ECs, P-(F56)-DOX and P-(Esbp)-DOX exhibited comparable efficacy in slowing the growth of primary 3LL tumors and prolonging the survival of these mice. Still, P-(Esbp)-DOX had more pronounced anti-tumor activity in mice bearing B16-F10 lung metastases after a single intravenous injection, at an equivalent DOX dose. Overall, our results indicate that the VEGFR-1- and E-selectin-targeted drug delivery systems evaluated here show enhanced anti-cancer activity, and prolonged the survival of mice after a single intravenous injection. This is thus the first study comparing the anti-tumor activity of VEGFR-1- and E-selectin-targeted polymer drug conjugates in the same TBM models at an equivalent drug dose.


Subject(s)
Acrylamides/administration & dosage , Antibiotics, Antineoplastic/administration & dosage , Doxorubicin/administration & dosage , Drug Carriers/administration & dosage , Vascular Endothelial Growth Factor Receptor-1/metabolism , Acrylamides/chemistry , Animals , Antibiotics, Antineoplastic/chemistry , Carcinoma, Lewis Lung/drug therapy , Carcinoma, Lewis Lung/metabolism , Carcinoma, Lewis Lung/pathology , Cell Line , Cell Survival/drug effects , Disease Models, Animal , Doxorubicin/chemistry , Drug Carriers/chemistry , E-Selectin/metabolism , HT29 Cells , Humans , Melanoma, Experimental/drug therapy , Melanoma, Experimental/metabolism , Mice, Inbred C57BL , Skin Neoplasms/drug therapy , Skin Neoplasms/metabolism , Treatment Outcome , Tumor Burden/drug effects
12.
Bioconjug Chem ; 27(4): 947-60, 2016 Apr 20.
Article in English | MEDLINE | ID: mdl-26974863

ABSTRACT

In recent years, siRNA technology has emerged as a promising strategy for gene silencing in cancer therapy. We have designed novel CD44-targeted polyion complexes (PICs) composed of poly(ethylene glycol)-block-polyethylenimine (PEG-b-PEI) and laminin-derived peptides (mA5G27D or mA5G27F) for in vivo siRNA delivery and gene silencing in tumors. The full-length A5G27 peptide (RLVSYNGIIFFLK), from which mA5G27D and mA5G27F are derived, binds to CD44v3 and CD44v6 and inhibits tumor cell migration, invasion, and angiogenesis. Thus, when attached to the surface of PICs, A5G27-based peptides can serve both as targeting ligands to navigate siRNA molecules directly to CD44-overexpressing tumors, and as anti-migratory agents to inhibit tumor progression. The mA5G27D- or mA5G27F-harboring PEG-b-PEI copolymers strongly condensed siRNA molecules into nanosized PICs presenting positive surface charges, low in vitro cytotoxicity, and high serum stability. mA5G27D- or mA5G27F-bearing PICs demonstrated high efficacy and selectivity in delivering siRAC1 into CD44-overexpressing cells, thereby silencing RAC1 mRNA and protein levels in such cells. These PICs presented substantial anti-migratory features in vitro and accumulated significantly in SK-OV-3 tumor-bearing mice, following 3 sequential intraperitoneal (i.p.) injections. Treatment of mice with 8 or 9 sequential parenteral (intravenous, (i.v.) or i.p.) injections of mA5G27F-PEG-b-PEI/siRNA efficiently inhibited tumor growth in two different CD44-overexpressing tumor mouse models (A549 and SK-OV-3), regardless of the type of siRNA (siPLK1 or siLUC) used. The results thus reveal the potential utility of this system for targeted delivery of siRNA molecules into solid tumors to prolong the survival time of mice, while at the same time reducing potential toxicity.


Subject(s)
Gene Expression , Hyaluronan Receptors/metabolism , Neoplasm Metastasis , Amino Acid Sequence , Animals , Cell Line, Tumor , Gene Silencing , Humans , Hyaluronan Receptors/chemistry , Mice , Oligopeptides/chemistry , Proton Magnetic Resonance Spectroscopy
13.
J Control Release ; 217: 102-12, 2015 Nov 10.
Article in English | MEDLINE | ID: mdl-26297207

ABSTRACT

There is currently no effective means to prevent or control metastatic dissemination of cancer cells. E-selectin, an adhesion molecule expressed exclusively on inflamed and angiogenic blood vessels, plays an important role in several rate-limiting steps of cancer metastasis. In this study, we assessed the in vivo antitumor efficacy of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers conjugated to an E-selectin binding peptide (Esbp, DITWDQLWDLMK) and equipped with the chemotherapeutic drug doxorubicin (P-(Esbp)-DOX) or with the proapoptotic peptide D(KLAKLAK)2 (P-(Esbp)-KLAK). Following a single intravenous injection, P-(Esbp)-DOX reduced tumor growth rate and prolonged the survival of mice bearing primary Lewis lung carcinoma (3LL) tumors significantly more than treatment with a non-targeted copolymer (P-DOX) or with free DOX. In an experimental B16-F10 lung metastasis model, a single intravenous dose of P-(Esbp)-DOX or P-(Esbp)-KLAK prolonged mice survival time significantly more than the non-targeted copolymers or the free drugs, and the percentage of complete tumor regression increased with increasing doses and with dosing frequency. In addition, mice pretreated with an E-selectin-targeted "drug-free" copolymer (P-(Esbp)-FITC) exhibited significantly fewer B16-F10 tumor foci in the lungs as compared with non-treated mice, demonstrating the anti-metastatic properties of the copolymer and its ability to control cancer spread through E-selectin-mediated interactions. Biodistribution analysis further confirmed the preferential accumulation of the E-selectin-targeted near-infrared fluorescently-labeled copolymer P-(Esbp)-IR783 in B16-F10 lung metastases. Taken together, this study demonstrates, for the first time, that the E-selectin targeted copolymer-drug conjugates can inhibit primary tumor growth and prevent metastases in vivo.


Subject(s)
Antineoplastic Agents/therapeutic use , Doxorubicin/therapeutic use , E-Selectin/metabolism , Neoplasms/drug therapy , Peptides/therapeutic use , Polymers/therapeutic use , Acrylamides/chemistry , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Cell Line , Cell Line, Tumor , Doxorubicin/chemistry , Doxorubicin/pharmacokinetics , Female , Melanoma, Experimental , Mice , Mice, Inbred C57BL , Neoplasms/metabolism , Neoplasms/pathology , Peptides/chemistry , Peptides/pharmacokinetics , Polymers/chemistry , Polymers/pharmacokinetics , Tumor Burden/drug effects
14.
Bioconjug Chem ; 26(2): 213-24, 2015 Feb 18.
Article in English | MEDLINE | ID: mdl-25560976

ABSTRACT

Dendritic cells (DCs) are a family of specialized antigen presenting cells (APCs) that detect antigens and initiate a wide spectrum of immune responses against them. These characteristics make them promising candidates for immunotherapy manipulations. In this study we designed and synthesized DC-targeted block copolymers composed of linear polyethylenimine (PEI) conjugated to hydrophilic polyethylene glycol (PEG) installed with a DC-targeting peptide (DC3, primary sequence FYPSYHSTPQRP). Two different conjugation procedures (basic and modified) were employed to synthesize the DC3-PEG-b-PEI and the control SCRM-PEG-b-PEI (with a scrambled DC3 peptide sequence) by one-pot synthesis, in two steps. In the first, basic conjugation procedure, PEG with N-hydroxysuccinimide (NHS) ester and maleimide (MAL) groups (NHS-PEG-MAL, 3.5 kDa) was first coupled to linear PEI (25 kDa) via the NHS group to yield the intermediate MAL-PEG-b-PEI, that was then conjugated to N-terminus-cysteine harboring peptides DC3 or SCRM via the MAL double bond to yield the final DC3-PEG-b-PEI or SCRM-PEG-b-PEI copolymers, respectively. In the second, modified conjugation procedure, Fmoc-cysteine harboring DC3 or SCRM peptides were first conjugated to NHS-PEG-MAL via the MAL group followed by coupling to linear PEI via the NHS functional group. Fmoc cleavage yielded the same final product as in the basic procedure. The modified conjugation procedure was capable of yielding block copolymers richer with peptides, as determined by (1)H NMR analysis. Self-assembly of DC3-PEG-b-PEI copolymers and DNA molecules yielded nanosized polyion complexes (polyplexes), with lower surface charge and limited cytotoxicity when compared to the PEI building block. Significant transfection efficiency of the DC-targeted polyplexes by murine dendritic DC2.4 cells was observed only in DC3-PEG-b-PEI/DNA polyplexes synthesized by the modified conjugation procedure. These polyplexes, with higher peptide-load, showed greater transfection capability in DC2.4 cells relative to the control nontargeted SCRM-PEG-b-PEI/DNA polyplexes, but not in endothelial cells. The transfection efficiency was comparable to or higher than that of the PEI/DNA positive control. The results indicate that PEGylated-PEI polyplexes show significant transfection efficiency into DCs when decorated with DC3 peptide, and are attractive candidates for immunotherapy via DCs.


Subject(s)
DNA/administration & dosage , Dendritic Cells/metabolism , Peptides/chemistry , Polyethylene Glycols/chemistry , Polyethyleneimine/chemistry , Transfection , Amino Acid Sequence , Animals , Cell Line , DNA/genetics , Maleimides/chemistry , Maleimides/metabolism , Mice , Peptides/metabolism , Polyethylene Glycols/metabolism , Polyethyleneimine/metabolism , Succinimides/chemistry , Succinimides/metabolism
15.
Mol Pharm ; 12(2): 453-62, 2015 Feb 02.
Article in English | MEDLINE | ID: mdl-25531245

ABSTRACT

Dendritic cells (DCs) possess unique abilities in initiating primary immune responses and thus represent prime targets for DNA-based vaccinations. Here, we describe the design and synthesis of mannosylated polyion complexes (PICs) composed of cationic polyethylenimine (PEI) and hydrophilic polyethylene glycol (PEG) segments, and bearing mono- and trivalent mannose as a ligand for targeting mannose receptor (MR/CD206)-positive DCs. Amino-terminated mannose (Man)-containing ligands in mono- and trivalent presentations (Man- and Man3-, respectively) were prepared and conjugated to PEG via an N-hydroxysuccinimide (NHS)-activated terminal. Thiolated PEI was conjugated to the mannosylated PEG via the maleimide (MAL)-activated terminal. The resulting positively charged diblock copolymers bearing mannoses (Man-PEG-b-PEI and Man3-PEG-b-PEI) were self-assembled with DNA to form PICs with lower surface charge than did their PEI building block and mean hydrodynamic diameters in the range of 100-450 nm, depending on the N/P ratio. Man3-PEG-b-PEI demonstrated a 3-4-fold greater transfection efficiency in MR-positive dendritic cell lines (THP-1, DC2.4), relative to Man-PEG-b-PEI, exhibited low cytotoxicity when compared with PEI, and showed low transfection efficiency in nondendritic HeLa cells. In preliminary in vivo experiments, Man-PEG-b-PEI/DNA and Man3-PEG-b-PEI/DNA demonstrated 2-3-fold higher gene delivery efficiency into CD11c(+) DCs collected from inguinal lymph nodes of C57/BL6 mice, when compared to PEI/DNA complexes, as shown by GFP expression measurements, 24 h post subcutaneous injection. The results indicate that the mannosylated PICs are a safe and effective gene delivery system, showing in vivo specificity toward CD11c(+) DCs.


Subject(s)
CD11c Antigen/genetics , Dendritic Cells/cytology , Gene Transfer Techniques , Polymers/chemistry , Transfection/methods , Animals , Cell Line , HeLa Cells , Humans , Mannose/chemistry , Mice , Polyethylene Glycols/chemistry , Polyethyleneimine/chemistry
16.
J Control Release ; 192: 182-91, 2014 Oct 28.
Article in English | MEDLINE | ID: mdl-25008468

ABSTRACT

There is increasing interest in the use of nanoparticle imaging probes for cancer diagnosis. However, various biological barriers limit the efficient delivery of nanoparticles to tumors following parenteral administration. We have investigated the applicability of a water-soluble polymeric imaging probe for improving the detection of gastrointestinal (GI) tumors after intra-luminal (colonic) administration. N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers bearing either fluorescein-isothiocyanate (FITC) or near-infrared fluorescence (NIRF) dye (IR-783) were conjugated with EPPT1 peptide, derived from the CDR3 Vh region of a monoclonal antibody (ASM2) raised against human epithelial cancer cells, for targeting under-glycosylated mucin-1 (uMUC-1) expressed in neoplastic tissues. The targeted FITC-labeled copolymer, P-(EPPT1)-FITC, was investigated for its ability to bind human CRC cells and tissue specimens in vitro. The uMUC-1-targeted NIRF-labeled copolymer, P-(EPPT1)-IR783, was assessed for its ability to detect colonic lesions in vivo. P-(EPPT1)-FITC demonstrated superior binding to colorectal cancer (CRC) cells that over-express the uMUC-1 antigen and exhibited selectivity towards human CRC tissue specimens, as compared to adjacent normal tissues from the same patient. When applied intra-colonically, P-(EPPT1)-IR783 significantly accumulated in cancerous tissue, relative to the adjacent normal mucosa of HT29 and LS174T tumor-bearing mice, and demonstrated higher signal intensities in colonic tumors, as compared to the non-targeted P-(GG-OH)-IR783 probe (i.e., without EPPT1). We found that P-(GG-OH)-IR783 can also accumulate specifically at tumor sites. The cancer-specific uptake and retention of P-(GG-OH)-IR783 was not mediated by organic anion transporting peptides (OATPs). Our findings indicate that the polymer-bound NIRF probe can successfully detect solid tumors in the GI tract following intra-colonic administration, and could be used in conjunction with colonoscopic procedures to improve the sensitivity of colonoscopies for polyp detection.


Subject(s)
Acrylamides , Colon/pathology , Colonoscopy/methods , Colorectal Neoplasms/diagnosis , Fluorescent Dyes , Mucin-1/metabolism , Peptides , Acrylamides/administration & dosage , Acrylamides/chemistry , Amino Acid Sequence , Animals , Cell Line, Tumor , Colon/metabolism , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Drug Delivery Systems , Female , Fluorescent Dyes/administration & dosage , Fluorescent Dyes/chemistry , Humans , Mice , Mice, Nude , Molecular Sequence Data , Optical Imaging , Peptides/administration & dosage , Peptides/chemistry , Peptides/metabolism , Rectum/metabolism , Rectum/pathology
17.
Pharm Res ; 31(3): 768-79, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24019024

ABSTRACT

PURPOSE: Cell penetrating peptides (CPPs) can mediate effective delivery of their associated drugs and drug carriers intracellularly, however their lack of cell specificity remains a major obstacle for their clinical development. We aimed at improving the cell specificity and therapeutic efficacy of HPMA copolymer-octaarginine (R8) conjugate (P-R8) in cells at the tumor micro-environment. METHODS: To avoid premature cell-penetration, the positively charged R8 moieties were masked via electrostatic complexation with various polyanionic molecules (heparin sulfate, hyaluronic acid, fucoidan and poly-glutamic acid). We followed the kinetics of the FITC-labeled P-R8 penetration into endothelial and cancer cells over-time after its complexation in vitro and further tested whether the in situ addition of a stronger polycation can trigger the release of P-R8 from the complexes to resume cell penetration activity. A murine model of B16-F10 lung metastasis was then used as an in vivo model for assessing the therapeutic efficacy of the P-R8, loaded with doxorubicin (P-R8-DOX), after its complexation with PGA. RESULTS: The intracellular penetration of P-R8-FITC was reversibly inhibited by forming electrostatic interactions with counter polyanions, and can be restored either gradually over time by dissociation from the polyanions, or promptly following the addition of protamine sulfate. Mice injected with B16-F10 cells and treated with P-R8-DOX/PGA complexes, exhibited a significant prolonged survival times when compared with DOX-treated mice or relative to mice treated with either P-R8-DOX or P-DOX alone. CONCLUSIONS: The gradual release of P-R8 from P-R8-DOX/PGA may improve the therapeutic efficacy of water-soluble based nanomedicines for the treatment of solid lung tumors.


Subject(s)
Antibiotics, Antineoplastic/administration & dosage , Cell-Penetrating Peptides/chemistry , Doxorubicin/administration & dosage , Drug Carriers/chemistry , Methacrylates/chemistry , Oligopeptides/chemistry , Animals , Antibiotics, Antineoplastic/therapeutic use , Cell Line, Tumor , Cell-Penetrating Peptides/metabolism , Doxorubicin/therapeutic use , Drug Carriers/metabolism , Lung/drug effects , Lung/pathology , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Methacrylates/metabolism , Mice , Mice, Inbred C57BL , Oligopeptides/metabolism
18.
Pharm Res ; 29(4): 1121-33, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22350800

ABSTRACT

PURPOSE: To evaluate the effect of the size of low molecular weight hyaluronan (LMW-HA) oligomers on the targeting ability of the HA-containing copolymers to the CD44-overexpressing cells for delivering Paclitaxel (PTX) to ovarian cancer. METHODS: LMW-HA oligosaccharides of 4, 6, 8, 10, 12 and 14 sugar residues were attained by digestion of HMW-HA using hyaluronate lyase at different incubation times and then attached to FITC-labeled HPMA copolymer precursor. The binding and uptake of the HA-modified HPMA-copolymer into CD44-expressing cells was studied by flow cytometry and confocal microscopy. PTX was further attached to HPMA-copolymer precursor bearing HA oligosaccharide at the size of 34 monosaccharides, through an acid-sensitive hydrazone linker. The cytotoxicity of the polymer was tested using cell viability assay. RESULTS: Polymer conjugates bearing HA oligomers at the size of 10 oligosaccharides and above (HA(10-14)) bind actively and profoundly to CD44-overexpressing ovarian cancer cells (SK-OV-3) and internalize to the greatest extent relative to HA-polymer conjugates of 8 oligomers and below (HA(4-8)). The HA-modified HPMA-copolymer PTX conjugate (P-(HA)(34)-PTX) exhibited 50-times higher cytotoxicity towards CD44-overexpressing cells relative to the control, non-targeted, HPMA-copolymer PTX conjugate (P-PTX). CONCLUSIONS: P-(HA)(34)-PTX was significantly more toxic than the non-targeted P-PTX in cells expressing high levels of CD44.


Subject(s)
Hyaluronan Receptors/biosynthesis , Hyaluronic Acid/administration & dosage , Hyaluronic Acid/chemistry , Ovarian Neoplasms/drug therapy , Paclitaxel/administration & dosage , Paclitaxel/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Drug Delivery Systems/methods , Female , Humans , Hyaluronan Receptors/genetics , Male , Oligosaccharides/administration & dosage , Oligosaccharides/chemistry , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Polymers/administration & dosage , Polymers/chemistry , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Tumor Cells, Cultured
19.
J Drug Target ; 19(10): 933-43, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22074249

ABSTRACT

Synthetic oligopeptides have emerged as a promising class of targeting ligands, providing a variety of choices for the construction of conjugates for desired ligand functionality. To explore the potential of short peptides as ligands for targeted delivery of macromolecular therapeutics for colorectal cancer (CRC), fluorescently labelled HPMA copolymers--bearing either G3-C12 or GE11 for targeting galectin-3 and epidermal growth factor receptor (EGFR), respectively--were synthesised and the mechanisms of their internalisation and subcellular fate in CRC cells were studied. The targetability of the G3-C12 bearing copolymers towards galectin-3 was further compared to that of galactose-containing copolymers. The resulting G3-C12-bearing conjugate actively and selectively targets CRC tumour cells over-expressing galectin-3 and exhibits superior targetability to galectin-3 when compared to the galactose-bearing copolymer. GE11 copolymer conjugate binds specifically and efficiently to EGFR over-expressing cells, thus mediating internalisation to a significantly higher extent relative the copolymer conjugated to a scrambled sequence peptide. We further incorporated doxorubicin (DOX) into GE11 bearing copolymer via an acid-labile hydrazone bond. The GE11-DOX copolymer conjugate demonstrated higher cytotoxicity toward EGFR over-expressing cells relative to the control non-targeted DOX conjugate. Altogether, our results show a proof of principle for the selective delivery of DOX to the target CRC cells.


Subject(s)
Antibiotics, Antineoplastic/pharmacology , Colorectal Neoplasms/drug therapy , Doxorubicin/analogs & derivatives , Drug Delivery Systems , Polymethacrylic Acids/pharmacology , Antibiotics, Antineoplastic/administration & dosage , Antibiotics, Antineoplastic/metabolism , Cell Line, Tumor , Colorectal Neoplasms/pathology , Doxorubicin/administration & dosage , Doxorubicin/metabolism , Doxorubicin/pharmacology , ErbB Receptors/metabolism , Galectin 3/metabolism , Humans , Ligands , Polymethacrylic Acids/administration & dosage , Polymethacrylic Acids/metabolism , Protein Binding
20.
Biomaterials ; 32(5): 1377-86, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21074848

ABSTRACT

Cell-penetrating peptides (CPPs) can be used for intracellular delivery of a broad variety of cargoes, including various nanoparticulate pharmaceutical carriers. However, the cationic nature of all CPP sequences, and thus lack of cell specificity, limits their in vivo use for drug delivery applications. Here, we have devised and tested a strategy for site-specific delivery of dyes and drugs into cancer cells by using polymers bearing a light activated caged CPP (cCPP). The positive charge of Lys residues on the minimum sequence of the CPP penetratin ((52)RRMKWKK(58)) was masked with photo-cleavable groups to minimize non-specific adsorption and cellular uptake. Once illuminated by UV light, these protecting groups were cleaved, the positively charged CPP regained its activity and facilitated rapid intracellular delivery of the polymer-dye or polymer-drug conjugates into cancer cells. We have found that a 10-min light illumination time was sufficient to enhance the penetration of the polymer-CPP conjugates bearing the proapoptotic peptide, (D)(KLAKLAK)(2), into 80% of the target cells, and to promote a 'switch' like cytotoxic activity resulting a shift from 100% to 10% in cell viability after 2 h. This report provides an example for tumor targeting by means of light activation of cell-penetrating peptides for intracellular drug delivery.


Subject(s)
Drug Delivery Systems , Light , Polymers/chemistry , Cell Line, Tumor , Cell-Penetrating Peptides/chemistry , Humans , Methacrylates/chemistry , Polymers/chemical synthesis
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