Anticancer therapeutics: targeting macromolecules and nanocarriers to hyaluronan or CD44, a hyaluronan receptor

The complex system involved in the synthesis, degradation and binding of the high molecular weight glycosaminoglycan hyaluronic acid (hyaluronan or HA) provides a variety of structures that can be exploited for targeted cancer therapy. In many cancers of epithelial origin there is an upregulation of CD44, a receptor that binds HA. In other cancers, HA in the tumor matrix is overexpressed. Both CD44 on cancer cells and HA in the matrix have been targets for anticancer therapy. Even though CD44 is expressed in normal epithelial cells and HA is part of the matrix of normal tissues, selective targeting to cancer is possible. This is because macromolecular carriers predominantly extravasate into the tumor and not normal tissue; thus CD44-HA targeted carriers administered intravenously localize preferentially into tumors. Anti-CD44 antibodies have been used in patients to deliver radioisotopes or mertansine for treatment of CD44 expressing tumors. In early phase clinical trials, patients with breast or head and neck tumors treated with anti-CD44 conjugates experienced stabilized disease. A dose-limiting toxicity was associated with distribution of the antibody-drug conjugate to the skin, a site in the body with a high level of CD44. HA has been used as a drug carrier and a ligand on liposomes or nanoparticles to target drugs to CD44 overexpressing cells. Drugs can be attached to HA via the carboxylate on the glucuronic acid residue, the hydroxyl on the N-acetylglucosamine or the reducing end which are located on a repeating disaccharide. Drugs delivered in HA-modified liposomes exhibited excellent antitumor activity both in vitro and in murine tumor models. The HA matrix is also a potential target for anticancer therapies. By manipulating the interaction of HA with cell surface receptors, either by degrading it with hyaluronidase or by interfering with CD44-HA interactions using soluble CD44 proteins, tumor progression was blocked. Finally, cytotoxic drugs or prodrug converting enzymes can be attached to the HA matrix to generate a cytotoxic fence around the tumor. This review describes how the complex interplay among cancer biology, the CD44-HA interaction, drug carriers and drug targeting has been used to improve anticancer therapies. As these approaches evolve, they hold forth the prospect of significantly improved targeted anticancer treatments.     

条件性重编程技术分离培养人原代乳腺癌干细胞

目的 应用条件性重编程细胞(Conditionally Reprogrammed Cells,CRCs)技术建立高效、稳定的人乳腺癌千细胞体外培养,建立潜在的乳腺癌干细胞靶向药研究模型.方法 收集人乳腺癌组织样本,采用CRCs技术分离培养原代乳腺癌细胞,用CD24免疫磁珠标记原代乳腺癌细胞后使用磁性细胞分选仪分选出乳腺...     

High Levels of Hyaluronic Acid Synthase-2 Mediate NRF2-Driven Chemoresistance in Breast Cancer Cells

Hyaluronic acid (HA), a ligand of CD44, accumulates in some types of tumors and is responsible for tumor progression. The nuclear factor erythroid 2-like 2 (NRF2) regulates cytoprotective genes and drug transporters, which promotes therapy resistance in tumors. Previously, we showed that high levels of CD44 are associated with NRF2 activation in cancer stem like-cells. Herein, we demonstrate that HA production was increased in doxorubicin-resistant breast cancer MCF7 cells (MCF7-DR) via the upregulation of HA synthase-2 (HAS2). HA incubation increased NRF2, aldo-keto reductase 1C1 (AKR1C1), and multidrug resistance gene 1 (MDR1) levels. Silencing of HAS2 or CD44 suppressed NRF2 signaling in MCF7-DR, which was accompanied by increased doxorubicin sensitivity. The treatment with a HAS2 inhibitor, 4-methylumbelliferone (4-MU), decreased NRF2, AKR1C1, and MDR1 levels in MCF7-DR. Subsequently, 4-MU treatment inhibited sphere formation and doxorubicin resistance in MCF7-DR. The Cancer Genome Atlas (TCGA) data analysis across 32 types of tumors indicates the amplification of HAS2 gene is a common genetic alteration and is negatively correlated with the overall survival rate. In addition, high HAS2 mRNA levels are associated with increased NRF2 signaling and poor clinical outcome in breast cancer patients. Collectively, these indicate that HAS2 elevation contributes to chemoresistance and sphere formation capacity of drug-resistant MCF7 cells by activating CD44/NRF2 signaling, suggesting a potential benefit of HAS2 inhibition.     

Spacer length impacts the efficacy of targeted docetaxel conjugates in prostate-specific membrane antigen expressing prostate cancer

Abstract

Combination of targeted delivery and controlled release is a powerful technique for cancer treatment. In this paper, we describe the design, synthesis, structure validation and biological properties of targeted and non-targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-docetaxel conjugates. Docetaxel (DTX) was conjugated to HPMA copolymer via a tetrapeptide spacer (–GFLG-). 3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA) was used as the targeting moiety to actively deliver DTX for treatment of Prostate-Specific Membrane Antigen (PSMA) expressing prostate cancer. Short and long spacer DUPA monomers were prepared, and four HPMA copolymer – DTX conjugates (non-targeted, two targeted with short spacer of different molecular weight and targeted with long spacer) were prepared via Reversible Addition-Fragmentation Chain Transfer (RAFT) copolymerization. Following confirmation of PSMA expression on C4-2 cell line, the DTX conjugates’ in vitro cytotoxicity was tested against C4-2 tumor cells and their anticancer efficacies were assessed in nude mice bearing s.c. human prostate adenocarcinoma C4-2 xenografts. The in vivo results show that the spacer length between targeting moieties and HPMA copolymer backbone can significantly affect the treatment efficacy of DTX conjugates against C4-2 tumor bearing nu/nu mice. Moreover, histological analysis indicated that the DUPA-targeted DTX conjugate with longer spacer had no toxicity in major organs of treated mice.     

CD44-tropic polymeric nanocarrier for breast cancer targeted rapamycin chemotherapy

In contrast with the conventional targeting of nanoparticles to cancer cells with antibody or peptide conjugates, a hyaluronic acid (HA) matrix nanoparticle with intrinsic-CD44-tropism was developed to deliver rapamycin for localized CD44-positive breast cancer treatment. Rapamycin was chemically conjugated to the particle surface via a novel sustained-release linker, 3-amino-4-methoxy-benzoic acid. The release of the drug from the HA nanoparticle was improved by 42-fold compared to HA-temsirolimus in buffered saline. In CD44-positive MDA-MB-468 cells, using HA as drug delivery carrier, the cell viability was significantly decreased compared to free rapamycin and CD44-blocked controls. Rat pharmacokinetics showed that the area under the curve of HA nanoparticle formulation was 2.96-fold greater than that of the free drug, and the concomitant total body clearance was 8.82-fold slower. Moreover, in immunocompetent BALB/c mice bearing CD44-positive 4T1.2neu breast cancer, the rapamycin-loaded HA particles significantly improved animal survival, suppressed tumor growth and reduced the prevalence of lung metastasis.From the Clinical EditorThis study demonstrates increased efficiency of rapamycin delivery and consequential treatment effects in a breast cancer model by hyaluronic acid - L-rapamycin conjugates with intrinsic tropism for CD44-positive cells.     

Biotin/Folate‐decorated Human Serum Albumin Nanoparticles of Docetaxel: Comparison of Chemically Conjugated Nanostructures and Physically Loaded Nanoparticles for Targeting of Breast Cancer

Docetaxel (DTX) is a widely used chemotherapeutic agent with very low water solubility. Conjugation of DTX to human serum albumin (HSA) is an effective way to increase its water solubility. Attachment of folic acid (FA) or biotin as targeting moieties to DTX‐HSA conjugates may lead to active targeting and specific uptake by cancer cells with overexpressed FA or biotin receptors. In this study, FA or biotin molecules were attached to DTX‐HSA conjugates by two different methods. In one method, FA or biotin molecules were attached to remaining NH₂ residues of HSA in DTX‐HSA conjugate by covalent bonds. In the second method, HSA‐FA or HSA‐biotin conjugates were synthesized separately and then combined by DTX‐HSA conjugate in proper ratio to prepare nanoparticles containing DTX‐HSA plus HSA‐FA or HSA‐biotin. Cell viability of different nanoparticle was evaluated on MDA‐MB‐231 (folate receptor positive), A549 (folate receptor negative), and 4T1 (biotin receptor positive) and showed superior cytotoxicity compared with free docetaxel (Taxotere^®). In vivo studies of DTX‐HSA‐FA and DTX‐HSA‐biotin conjugates in BULB/c mice, tumorized by 4T1 cell line, showed the conjugates prepared in this study were more powerful in the reduction in tumor size and increasing the survival rate when compared to free docetaxel.     

Uptake and Cytotoxicity of Docetaxel-Loaded Hyaluronic Acid-Grafted Oily Core Nanocapsules in MDA-MB 231 Cancer Cells

Purpose

It is hypothesized that docetaxel (Doc)-loaded hyaluronic acid (HA)-polyethylene glycol/poly(ε-caprolactone)-grafted oily core nanocapsules (NCs) can enhance the drug cytotoxicity and uptake in CD44 expressing breast cancer (BC) cells (MDA-MB 231).

Methods

NCs were prepared, optimized and characterized by dynamic light scattering, transmission electron microscopy (TEM), and powder X-ray diffraction (PXRD). In vitro cytotoxicity tests [MTS, level of reactive oxygen species (ROS) and level of reduced glutathione (GSH)] were performed in BC cells. The contribution of CD44 to the NCs cellular uptake was elucidated using an anti CD44 antibody blockage and a CD44 negative NIH3T3 cell line.

Results

The optimum formulation of Doc-loaded HA oily core NCs had respective mean diameter, polydispersity, and drug encapsulation efficiency of 224.18 nm, 0.32, and 60.38%. The NCs appeared spherical with low drug crystallinity, while the drug release data fitted to first order equation. Compared to that of ungrafted NCs, the cytotoxicity of Doc-loaded HA-grafted NCs was significantly enhanced (p<0.05). A decrease of the intracellular level of ROS was reversely correlated with that of GSH. Interestingly, the cellular internalization of HA-grafted NCs mediated CD44 was dramatically enhanced (3 to 4-fold) with respect to the absence of specific biomarker or targeting ligand.

Conclusions

The use of HA-grafted NCs enhanced the selective drug payload, cytotoxicity and uptake in MDA-MB 231 cells. Therefore, it could be a promising template for safe and effective delivery of Doc and similar chemotherapeutic agents in cancer cells.     

Doses and Time‐Dependent Effects of 3′‐Azido‐3′‐deoxythymidine on T47D Human Breast Cancer Cells in vitro

The objective of the present work was to study the effects of 3′‐azido‐3′‐deoxythymidine (azidothymidine, Zidovudine^®) on human breast cancer cells by using, as a model, the T47D cell line (typified as oestrogen‐dependent and p53‐mutated). Low azidothymidine doses (3.125 μM) increase the percentage of cells in S‐phase, with the effect reversing after 24 hr of incubation; as azidothymidine doses increase, the magnitude and duration of its effect increase proportionally, although, even with the highest concentrations (50–100 μM) the effects decline after 48 hr of incubation. If media (containing azidothymidine or vehicle) are daily renewed, the azidothymidine effects (accumulation of cells in S‐phase) are higher and decline later than when media and drug are not changed during the whole culture period, thereby suggesting that the reversion of azidothymidine effects could be related with a degradation of the drug or accumulation in media of substances which counteract its effects. Azidothymidine inhibits T47D cell proliferation at concentrations higher than 50 μM. The exposure to 50 or 100 μM azidothymidine induced cell apoptosis after 48 hr or more of incubation. We conclude that: a) azidothymidine, with appropriate doses and duration of treatment, synchronizes cells in S‐phase, inhibits proliferation, and induces apoptosis, b) the discontinuous application of the drug rather than continuous exposure to it increases its efficiency to synchronize the T47D cell cycle. This in vitro anti‐breast cancer activity suggests that a possible clinical usefulness of azidothymidine, either alone or associated with other drugs with cycle‐specific antitumoural activity circumscribed to the S‐phase of cell cycle, is worthy of investigation.     

Overcoming vincristine resistance in cancer: Computational design and discovery of piperine‐inspired P‐glycoprotein inhibitors

P‐glycoprotein (P‐gp)/MDR‐1 plays a major role in the development of multidrug resistance (MDR) by pumping the chemotherapeutic drugs out of the cancer cells and reducing their efficacy. A number of P‐gp inhibitors were reported to reverse the MDR when co‐administered with chemotherapeutic drugs. Unfortunately, none has approved for clinical use due to toxicity issues. Some of the P‐gp inhibitors tested in the clinics are reported to have cross‐reactivity with CYP450 drug‐metabolizing enzymes, resulting in unpredictable pharmacokinetics and toxicity of co‐administered chemotherapeutic drugs. In this study, two piperine analogs ( 3 and 4 ) having lower cross‐reactivity with CYP3A4 drug‐metabolizing enzyme are identified as P‐glycoprotein (P‐gp) inhibitors through computational design, followed by synthesis and testing in MDR cancer cell lines over‐expressing P‐gp (KB Ch^R 8–5, SW480‐VCR, and HCT‐15). Both the analogs significantly increased the vincristine efficacy in MDR cancer cell lines at low micromole concentrations. Specifically, 3 caused complete reversal of vincristine resistance in KB Ch^R 8–5 cells and found to act as competitive inhibitor of P‐gp as well as potentiated the vincristine‐induced NF‐KB‐mediated apoptosis. Therefore, 3 ((2E,4E)‐1‐(6,7‐dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)‐5‐(4‐hydroxy‐3‐methoxyphenyl)penta‐2,4‐dien‐1‐one) can serve as a potential P‐gp inhibitor for in vivo investigations, to reverse multidrug resistance in cancer.     

Targeted Delivery of Doxorubicin by HPMA Copolymer-Hyaluronan Bioconjugates

Purpose. Overexpression of hyaluronan (HA) receptors on cancer cells results in enhanced endocytotic uptake of the drug conjugate. An N-(2-hydroxypropyl)methacrylamide (HPMA)-HA polymeric drug delivery system was used for targeted delivery of doxorubicin to cancer cells. Methods. HA-doxorubicin (DOX) bioconjugates (HA-DOX), and HPMA copolymer-DOX conjugates containing HA as a side chain (HPMA-HA-DOX) were synthesized. The cytotoxicity of the polymer-drug conjugate was evaluated via in vitro cell culture. The internalization of the conjugate was visualized by fluorescence microscopy. Results. Cytotoxicity of HPMA-HA-DOX targeted bioconjugate was higher against human breast cancer (HBL-100), ovarian cancer (SKOV-3), and colon cancer (HCT-116) cells when compared to the non-targeted HPMA-DOX conjugate. Fluorescence confocal microscopy revealed that the targeted HPMA-HA-DOX conjugates were internalized more efficiently by cancer cells relative to the non-targeted HPMA-DOX conjugate. Both HPMA-DOX and HPMA-HA-DOX showed minimal cytotoxicity toward mouse fibroblast NIH 3T3 cells. The internalization of polymer conjugates was correlated with their cytotoxicity. Conclusions. Selective delivery of anti-cancer agents to cancer cells was achieved by biochemical targeting. The HA-modified HPMA copolymer showed improved toxicity due to receptor-mediated uptake of the macromolecular drug.     

Ablation of Breast Cancer Cells Using Trastuzumab‐Functionalized Multi‐Walled Carbon Nanotubes and Trastuzumab‐Diphtheria Toxin Conjugate

Trastuzumab (Herceptin^®) is a monoclonal antibody (mAb) for specific ablation of HER2‐overexpressing malignant breast cancer cells. Intensification of antiproliferative activity of trastuzumab through construction of immunotoxins and nano‐immunoconjugates is a promising approach for treatment of cancer. In this study, trastuzumab was directly conjugated to diphtheria toxin (DT). Also, conjugates of trastuzumab and multiwalled carbon nanotubes (MWCNT) were constructed by covalent immobilization of trastuzumab onto MWCNTs. Then, antiproliferative activity of the fusion constructs against HER2‐overexpressing SK‐BR‐3 and also HER2‐negative MCF‐7 cancer cell lines were examined. Cells treated with trastuzumab‐MWCNT conjugates were irradiated with near‐infrared (NIR) light. Efficient absorption of NIR radiation and its conversion to heat by MWCNTs can be resulted to thermal ablation of cancerous cells. Our results strongly showed that both trastuzumab‐MWCNT and trastuzumab‐DT conjugates were significantly efficient in the specific killing of SK‐BR‐3 cells. Targeting of MWCNTs to cancerous cells using trastuzumab followed by exposure of cells to NIR radiation was more efficient in repression of cell proliferation than treatment for cancer cells with trastuzumab‐DT. Our results also showed that conjugation linkers can significantly affect the cytotoxicity of MWCNT‐immunoconjugates. In conclusion, our data demonstrated that trastuzumab‐MWCNT is a promising nano‐immunoconjugate for killing of HER2‐overexpressing cancerous cells.     

Low‐dose metronomic chemotherapy with cisplatin enhanced immunity in a murine model of ectopic cervical cancer

Previous researchers have claimed that metronomic low‐dose/dense chemotherapy can enhance the therapeutic effectiveness of cisplatin treatment in the control of cancer. Therefore, the aim of this study was to explore the effectiveness of metronomic drug delivery with regards to its effects on adaptive immunity in a murine model of ectopic cervical cancer. The effectiveness of long‐term low‐dose/dense cisplatin treatment in HPV E7‐expressing TC‐1 cells was evaluated via morphological observations. Tumour mass and survival curves were used to determine the antitumour effect against E7‐expressing tumours. After experimental mice had been treated with low‐dose/dense cisplatin therapy, flow cytometry was used to measure the expression of MHC class I surface antigens on cultured TC‐1 cells. Splenocytes expressing both interferon (IFN)‐γ and CD8 responsible for E7 antigens and the T_reg population were also quantified using flow cytometry. The results indicate that in vivo treatment with metronomic cisplatin suppresses the growth of cultured TC‐1 cells. An increase was also observed in the number of splenocytes expressing both IFN‐γ and CD8 responsible for E7 antigens and the T_reg population. These results support previous reports that metronomic low‐dose/dense cisplatin chemotherapy is an effective treatment against ectopic cervical cancer with E7‐expression.     

Single‐walled carbon nanotube and graphene nanodelivery of gambogic acid increases its cytotoxicity in breast and pancreatic cancer cells

Graphene and single‐walled carbon nanotubes were used to deliver the natural low‐toxicity drug gambogic acid (GA) to breast and pancreatic cancer cells in vitro, and the effectiveness of this complex in suppressing cellular integrity was assessed. Cytotoxicity was assessed by measuring lactate dehydrogenase release, mitochondria dehydrogenase activity, mitochondrial membrane depolarization, DNA fragmentation, intracellular lipid content, and membrane permeability/caspase activity. The nanomaterials showed no toxicity at the concentrations used, and the antiproliferative effects of GA were significantly enhanced by nanodelivery. The results suggest that these complexes inhibit human breast and pancreatic cancer cells grown in vitro. This analysis represents a first step toward assessing their effectiveness in more complex, targeted, nanodelivery systems. Copyright © 2014 John Wiley & Sons, Ltd.     

Design,synthesis, and biological activity evaluation of campthothecin‐HAA‐Norcantharidin conjugates as antitumor agents in vitro

Three components of Camptothecin, hydroxyacetic acid, and functionalized norcantharidins were constructed together to form a novel series of camptothecin derivatives in a good yield. The synthesized campthothecin‐HAA‐norcantharidin conjugate pro‐drugs could suppress cancer cell growth in vitro. These conjugated pro‐drug molecules possess therapeutic potential as novel bi‐functional conjugates platforms for cancer treatment.     

Design,synthesis and biological evaluation of peptide‐NSAID conjugates for targeted cancer therapy

Linear arginine‐glycine‐aspartic acid (RGD) and asparagine‐glycine‐arginine (NGR) peptide‐nonsteroidal anti‐inflammatory drug conjugates were synthesized to evaluate their anticancer effect. Two well‐known targeting peptide sequences, RGD and NGR, were conjugated with naproxen and ibuprofen. It is expected that the RGD peptide selectively binds to α_v‐integrin receptors, which are highly expressed in cancer cells, and that the NGR peptide selectively targets aminopeptidase N (APN/CD13, EC 3.4.11.2), which is overexpressed in blood vessels of tumors. To investigate the impact of possible steric hindrance due to the attachment of the drug to the peptide, a linear six‐carbon linker (hexanoic acid) was also used as a spacer. Cytotoxic effects of the synthesized compounds were evaluated against several cancer cell lines, including MCF‐7, A2780 (α_vβ₃ positive), OVCAR3 (high α_vβ₃), HT‐1‐80, and SKOV‐3 cells (CD13 positive). The NGR conjugate forms of both ibuprofen and naproxen showed better activity against the SKOV‐3 tumor cell line. The improved binding of these conjugates to their receptors was confirmed by docking studies.     

Quantitative Relationships Between the Cytotoxicity of Flavonoids on the Human Breast Cancer Stem‐Like Cells MCF7‐SC and Their Structural Properties

As some breast cancer‐related deaths can be attributed to the metastasis of cancer stem cells, chemotherapeutic agents targeting breast cancer stem cells are of interest as a potential treatment. Flavonoids that exhibit cytotoxicity on breast cancer stem cells have rarely been observed. Thus, the objective of this study was to measure potential cytotoxic effects of 42 different flavonoids on the human breast cancer stem‐like cell line, MCF7‐SC. The relationship between flavonoid structural properties and cytotoxicity has not been reported previously; therefore, we determined quantitative structure–activity relationships using both comparative molecular field analysis and comparative molecular similarity analysis. Further biological experiments including Western blot analysis, flow cytometry, and immunofluorescence microscopy were also conducted on the most cytotoxic 8‐chloroflavanone.     

Enhanced Cytotoxicity to Cancer Cells by Codelivery and Controlled Release of Paclitaxel‐loaded Sirolimus‐conjugated Albumin Nanoparticles

Recently, it is suggested that mTOR signaling pathway is an important mediator in many cancers especially breast cancer. Therefore, effects of sirolimus as a mTOR inhibitor in breast cancer have been studied in combination with paclitaxel with or without controlled release effect. In this work, we prepared a water‐soluble formulation of sirolimus‐conjugated albumin nanoparticles loaded with paclitaxel, to study the effects of sirolimus concentration when it releases more later than paclitaxel in comparison with sirolimus–paclitaxel‐loaded albumin nanoparticles. Also effects of paclitaxel loading on cytotoxic properties of nanoparticles were studied. Sirolimus was succinylated at 42‐OH with enzymatic reaction of Candida antarctica lipase B, and then its carboxylic group was activated with EDC/NHS and conjugated to the lysine residues of albumin. Paclitaxel was loaded on albumin surface by nab technique in concentration range of 0–10 μg/mL. Sirolimus‐conjugated nanoparticles with 0.01 μg/mL paclitaxel showed lowest cell viability of 44% while it was 53% for non‐conjugated nanoparticles in MDA‐MB‐468 cell lines after 48 h (p‐value = 0.003). In MCF‐7 cell lines, sirolimus‐conjugated nanoparticles with 0.1 μg/mL paclitaxel showed lowest cell viability of 35.69% while it was 48% for non‐conjugated nanoparticles after 48 h (p‐value = 0.03). We guess that when cancer cell lines arrest in G2‐M by anticancer drugs like paclitaxel, Akt activates mTOR to make cells continue living, then inhibiting mTOR can enhance anticancer effects.     

Antibody‐Drug Conjugates: A Clinical Pharmacy Perspective on an Emerging Cancer Therapy

Antibody‐drug conjugates (ADCs) combine highly specific monoclonal antibodies with potent cytotoxic drugs. Their synergy allows for targeted delivery of toxic drugs to cancer cells while sparing systemic exposure. In this review, we focus on the history and clinical applications of ADCs approved by the U.S. Food and Drug Administration (FDA) for the treatment of cancer and highlight new ADCs in the drug development pipeline. Three ADCs have received FDA approval thus far. Gemtuzumab ozogamicin, although withdrawn from the U.S. market, may still be an effective treatment modality in subsets of patients with acute myeloid leukemia. Brentuximab vedotin and ado‐trastuzumab emtansine have shown improved efficacy and safety data compared with standard chemotherapy for the treatment of advanced lymphoma and breast cancer, respectively. With a number of ADCs with promising preliminary data in the clinical trial pipeline, cancer therapy is moving forward from traditional chemotherapy to targeted treatment modalities driven by the specificity of monoclonal antibodies and advancing biotechnology.     

Water-soluble polymers for targeted drug delivery to human squamous carcinoma of head and neck

Human squamous cell carcinoma of the head and neck (SCCHN) is characterized by over expression of a tumor cell surface-specific receptor namely Hsp47/CBP2 that makes it a favorable candidate for targeted delivery of anticancer drugs. Several synthetic peptides have been identified as effective ligands for binding to CBP2. The purpose of this study is to investigate the potential of water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) conjugates containing a Hsp47/CBP2 binding peptide sequence, namely WHYPWFQNWAMA for targeted delivery to SCCHN. An HPMA copolymer containing Dox and CBP2 targeting peptide conjugated via lysosomally degradable glycylphenylalanylleucylglycine (GFLG) spacer was synthesized by free radical precipitation copolymerization. A control polymer without targeting moiety was also synthesized. The conjugates were characterized for drug content, peptide content, molecular weight and molecular weight distribution. The uptake of polymeric conjugates by both drug resistant and drug sensitive SCCHN cells were determined in vitro by flow cytometry using FACS scan analysis. Cytotoxicity of the conjugates towards drug sensitive as well as multidrug resistant SCCHN cells were evaluated by a clonal survival assay and compared to free Dox. The cytotoxicity of the free peptide was similarly evaluated. The internalization and subcellular fate of the conjugates in drug sensitive SCCHN cells was monitored using confocal microscopy. The new targetable copolymer contained 0.16 mmole peptide/g polymer. Studies on drug sensitive SCCHN cells demonstrated lesser uptake of both targeted and non-targeted conjugates compared to free Dox suggesting a slower endocytic mechanism of uptake for the conjugates as opposed to rapid diffusion of free Dox. At higher Dox equivalent concentrations (>20 μM) the targeted conjugate showed significantly higher uptake (p≤0.028) than the non-targeted conjugate. The uptake of the targeted conjugate was inhibited in the presence of an anti Hsp47 antibody suggesting the involvement of active receptor mediated endocytosis in cell entry of the conjugate. Compared to free Dox, the targeted and non-targeted conjugates caused marginally lower inhibition (p≤0.01) of the drug sensitive SCCHN cells. In contrast, the same conjugates showed significantly higher uptake (p≤0.004) by drug resistant SCCHN cells and caused significantly higher inhibition (p≤0.02) of drug resistant SCCHN cells when compared to free Dox. Results suggest that the polymeric conjugates were able to overcome drug resistance. Confocal microscopy studies demonstrated the uptake of the polymeric conjugates, followed by internalization, intralysosomal localization and subsequent release of Dox. HPMA copolymer-Dox-peptide conjugates targeted to SCCHN cells were able to overcome drug resistance and increase efficacy in vitro. The results suggest that targetable polymeric conjugates have potential to improve systemic head and neck cancer chemotherapy by increasing tumor localization and reducing dose-limiting toxicity.