Search tips
Search criteria

Results 1-25 (26)

Clipboard (0)

Select a Filter Below

Year of Publication
Document Types
1.  Toxicity and efficacy evaluation of multiple targeted polymalic acid conjugates for triple-negative breast cancer treatment 
Journal of drug targeting  2013;21(10):956-967.
Engineered nanoparticles are widely used for delivery of drugs but frequently lack proof of safety for cancer patient's treatment. All-in-one covalent nanodrugs of the third generation have been synthesized based on a poly(β-L-malic acid) (PMLA) platform, targeting human triple-negative breast cancer (TNBC). They significantly inhibited tumor growth in nude mice by blocking synthesis of epidermal growth factor receptor, and α4 and β1 chains of laminin-411, the tumor vascular wall protein and angiogenesis marker. PMLA and nanodrug biocompatibility and toxicity at low and high dosages were evaluated in vitro and in vivo. The dual-action nanodrug and single-action precursor nanoconjugates were assessed under in vitro conditions and in vivo with multiple treatment regimens (6 and 12 treatments). The monitoring of TNBC treatment in vivo with different drugs included blood hematologic and immunologic analysis after multiple intravenous administrations. The present study demonstrates that the dual-action nanoconju-gate is highly effective in preclinical TNBC treatment without side effects, supported by hematologic and immunologic assays data. PMLA-based nanodrugs of the Polycefin™ family passed multiple toxicity and efficacy tests in vitro and in vivo on preclinical level and may prove to be optimized and efficacious for the treatment of cancer patients in the future.
PMCID: PMC4043297  PMID: 24032759
Hematologic; immunogenicity; in vivo treatment; nanoconjugate drugs; polymalic acid; toxicity; triple-negative breast cancer
Laminins are the major constituents of blood vessel basement membranes (BMs). Each laminin is a trimer consisting of three assembled polypeptide chains, α, β and γ. More than 15 laminin isoforms are known to date and the expression of specific isoforms may change in certain pathological conditions. Here we show that during progression of glial tumors laminin-9 (α4β2γ1) is switched to laminin-8 (α4β1γ1), which is dramatically increased in glial brain tumors. Laminin-8 overproduction by glial tumor cells facilitates spread of glioma. Brain tumors with laminin-8 overexpression recur faster after standard treatment and patients have shorter survival time. Laminin-8 may be thus used as a predictor of tumor recurrence, patient survival and as a potential molecular target for glioma therapy.
PMCID: PMC3506377  PMID: 16146715
Laminin-8; Laminin-9; Basement Membrane; Extracellular Matrix; Angiogenesis; Human; Cancer; Tumor; Neoplasm; Glioma; Glioblastoma Multiforme; Recurrence; Survival; Invasion; Morpholino antisense; Review
3.  Polymalic Acid–Based Nanobiopolymer Provides Efficient Systemic Breast Cancer Treatment by Inhibiting both HER2/neu Receptor Synthesis and Activity 
Cancer research  2011;71(4):1454-1464.
Biodegradable nanopolymers are believed to offer great potential in cancer therapy. Here, we report the characterization of a novel, targeted, nanobiopolymeric conjugate based on biodegradable, nontoxic, and nonimmunogenic PMLA [poly(β-l-malic acid)]. The PMLA nanoplatform was synthesized for repetitive systemic treatments of HER2/neu-positive human breast tumors in a xenogeneic mouse model. Various moieties were covalently attached to PMLA, including a combination of morpholino antisense oligonucleotides (AON) directed against HER2/neu mRNA, to block new HER2/neu receptor synthesis; anti-HER2/neu antibody trastuzumab (Herceptin), to target breast cancer cells and inhibit receptor activity simultaneously; and transferrin receptor antibody, to target the tumor vasculature and mediate delivery of the nanobiopolymer through the host endothelial system. The results of the study showed that the lead drug tested significantly inhibited the growth of HER2/neu-positive breast cancer cells in vitro and in vivo by enhanced apoptosis and inhibition of HER2/neu receptor signaling with suppression of Akt phosphorylation. In vivo imaging analysis and confocal microscopy demonstrated selective accumulation of the nanodrug in tumor cells via an active delivery mechanism. Systemic treatment of human breast tumor-bearing nude mice resulted in more than 90% inhibition of tumor growth and tumor regression, as compared with partial (50%) tumor growth inhibition in mice treated with trastuzumab or AON, either free or attached to PMLA. Our findings offer a preclinical proof of concept for use of the PMLA nanoplatform for combination cancer therapy.
PMCID: PMC3428373  PMID: 21303974
4.  Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine? 
Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.
PMCID: PMC4118553  PMID: 24962356
Chemistry; Issue 88; Cancer treatment; personalized medicine; polymalic acid; nanodrug; biopolymer; targeting; host compatibility; biodegradability
5.  The optimization of polymalic acid peptide copolymers for endosomolytic drug delivery 
Biomaterials  2011;32(22):5269-5278.
Membranolytic macromolecules are promising vehicles forcytoplasmic drug delivery, but their efficiency and safety remains primary concerns. To address those concerns, membranolytic properties of various poly(β-l-malic acid) (PMLA) copolymers were extensively investigated as a function of concentration and pH. PMLA, a naturally occurring biodegradable polymer, acquires membranolytic activities after substitution of pendant carboxylates with hydrophobic amino acid derivatives. Ruled by hydrophobization and charge neutralization, membranolysis of PMLA copolymers increased as a function of polymer molecular weight and demonstrated a maximum with 50% substitution of carboxylates. Charge neutralization was achieved either conditionally by pH-dependent protonation or permanently by masking carboxylates. Membranolysis of PMLA copolymers containing tripeptide ofleucine, tryptophan and phenylalanine were pH-dependent in contrast to pH-independent copolymers of Leucineethylester and Leu-Leu-Leu-NH2 with permanent charge neutralization. PMLA and tripeptides seemed a unique combination for pH-dependent membranolysis. In contrast to nontoxic pH-dependent PMLA copolymers, pH-independent copolymers were found toxic at high concentration, which is ascribed to their nonspecific disruption of plasma membrane at physiological pH.pH-dependent copolymers were membranolytically active only at acidic pH typical of maturating endosomes, and are thus devoid of cytotoxicity. The PMLA tripeptide copolymers are useful for safe and efficient cytoplasmic delivery routed through endosome.
PMCID: PMC4110056  PMID: 21514661
6.  Biocompatible nanopolymers: the next generation of breast cancer treatment? 
Nanomedicine (London, England)  2012;7(10):1467-1470.
PMCID: PMC4086916  PMID: 23148535
biomarkers; breast cancer; nanodrugs; nanopolymers; personalized medicine; polymalic acid
7.  Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine? 
Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.
PMCID: PMC4118553  PMID: 24962356
Chemistry; Issue 88; Cancer treatment; personalized medicine; polymalic acid; nanodrug; biopolymer; targeting; host compatibility; biodegradability
8.  Polymalic acid nanobioconjugate for simultaneous inhibition of tumor growth and immunostimulation in HER2/neu-positive breast cancer✩ 
Breast cancer remains the second leading cause of cancer death among women in the United States. The breast cancer prognosis is particularly poor in case of tumors overexpressing the oncoprotein HER2/neu. A new nanobioconjugate of the Polycefin family of anti-cancer drugs based on biodegradable and non-toxic polymalic acid (PMLA) was engineered for a multi-pronged attack on HER2/neu-positive breast cancer cells. An antibody cytokine fusion protein consisting of the immunostimulatory cytokine interleukin-2 (IL-2) genetically fused to an antibody specific for human HER2/neu [anti-HER2/neu IgG3-(IL-2)] was covalently attached to the PMLA backbone to target HER2/neu expressing tumors and ensuring the delivery of IL-2 to the tumor microenvironment. Antisense oligonucleotides (AON) were conjugated to the nanodrug to inhibit the expression of vascular tumor protein laminin-411 in order to block tumor angiogenesis. It is shown that the nanobioconjugate was capable of specifically binding human HER2/neu and retaining the biological activity of IL-2. We also showed the uptake of the nanobioconjugate by HER2/neu-positive breast cancer cells and enhanced tumor targeting in vivo. In addition, the nanobioconjugate was capable of eliciting anti-tumor activity in immunocompetent mice bearing D2F2/E2 murine mammary tumors that express human HER2/neu. Both IgG1 and IgG2a levels were significantly increased in animals treated with the PMLA-fusion nanobioconjugate compared to animals treated with the antibody–cytokine fusion protein alone or control animals, indicative of the induction of a humoral (TH2) and cell-mediated (TH1) immune responses. Animal survival in vivo was significantly longer after treatment with leading nanobioconjugate with fusion [anti-HER2/neu IgG3-(IL-2)] antibody, p < 0.05. The combination of these molecules on a single polymeric platform is expected to act through direct elimination of cancer cells, inhibition of tumor angiogenesis, and orchestration of a potent immune response against tumor.
PMCID: PMC3971991  PMID: 23770212
Polymalic acid; Nanobioconjugate; Nanopolymer; HER2/neu; Antibody fusion protein; IL-2; Laminin-411; Breast cancer
9.  Distinct mechanisms of membrane permeation induced by two polymalic acid copolymers 
Biomaterials  2012;34(1):217-225.
Anionic polymers are valuable components used in cosmetics and health sciences, especially in drug delivery, because of their chemical versatility and low toxicity. However, because of their highly negative charge they pose problems for penetration through hydrophobic barriers such as membranes. We have engineered anionic polymalic acid (PMLA) to penetrate biological membranes. PMLA copolymers of leucine ethyl ester (P/LOEt) or trileucine (P/LLL) show either pH-independent or pH-dependent activity for membrane penetration. We report here for the first time on the mechanisms which are different for those two copolymers. Formation of hydrophobic patches in either copolymer is detected by fluorescence techniques. The copolymers display distinctly different properties in solution and during membranolysis. P/LOEt copolymer binds to membrane as single molecules with high affinity, and induces leakage cooperatively through a mechanism known as “carpet” model, in which the polymer aligns at the surface throughout the entire process of membrane permeation. In contrast, P/LLL self-assembles to form an oligomer of 105 nm in a pH-dependent manner (pKa 5.5) and induces membrane leakage through a two-phase process: the concentration dependent first-phase of insertion of the oligomer into membrane followed by a concentration independent second-phase of rearrangement of the membrane-oligomer complex. The insertion of P/LLL is facilitated by hydrophobic interactions between trileucine side chains and lipids in the membrane core, resulting in transmembrane pores, through mechanism known as “barrel-stave” model. The understanding of the mechanism paves the way for future engineering of polymeric delivery systems with optimal cytoplasmic delivery efficiency and reduced systemic toxicity.
PMCID: PMC3487713  PMID: 23063368
10.  Transferrin receptors and the targeted delivery of therapeutic agents against cancer 
Biochimica et biophysica acta  2011;1820(3):291-317.
Traditional cancer therapy can be successful in destroying tumors, but can also cause dangerous side effects. Therefore, many targeted therapies are in development. The transferrin receptor (TfR) functions in cellular iron uptake through its interaction with transferrin. This receptor is an attractive molecule for the targeted therapy of cancer since it is upregulated on the surface of many cancer types and is efficiently internalized. This receptor can be targeted in two ways: 1) for the delivery of therapeutic molecules into malignant cells or 2) to block the natural function of the receptor leading directly to cancer cell death.
Scope of review
In the present article we discuss the strategies used to target the TfR for the delivery of therapeutic agents into cancer cells. We provide a summary of the vast types of anti-cancer drugs that have been delivered into cancer cells employing a variety of receptor binding molecules including Tf, anti-TfR antibodies, or TfR-binding peptides alone or in combination with carrier molecules including nanoparticles and viruses.
Major conclusions
Targeting the TfR has been shown to be effective in delivering many different therapeutic agents and causing cytotoxic effects in cancer cells in vitro and in vivo.
General significance
The extensive use of TfR for targeted therapy attests to the versatility of targeting this receptor for therapeutic purposes against malignant cells. More advances in this area are expected to further improve the therapeutic potential of targeting the TfR for cancer therapy leading to an increase in the number of clinical trials of molecules targeting this receptor.
PMCID: PMC3500658  PMID: 21851850
transferrin receptor; CD71; cancer; nanoparticles; immunotoxins; delivery; conjugates; gene therapy
11.  Nanoplatforms for constructing new approaches to cancer treatment, imaging, and drug delivery: What should be the policy? 
NeuroImage  2010;54(Suppl 1):S106-S124.
Nanotechnology is the design and assembly of submicroscopic devices called nanoparticles, which are 1–100 nm in diameter. Nanomedicine is the application of nanotechnology for the diagnosis and treatment of human disease. Disease-specific receptors on the surface of cells provide useful targets for nanoparticles. Because nanoparticles can be engineered from components that (1) recognize disease at the cellular level, (2) are visible on imaging studies, and (3) deliver therapeutic compounds, nanotechnology is well suited for the diagnosis and treatment of a variety of diseases. Nanotechnology will enable earlier detection and treatment of diseases that are best treated in their initial stages, such as cancer. Advances in nanotechnology will also spur the discovery of new methods for delivery of therapeutic compounds, including genes and proteins, to diseased tissue. A myriad of nanostructured drugs with effective site-targeting can be developed by combining a diverse selection of targeting, diagnostic, and therapeutic components. Incorporating immune target specificity with nanostructures introduces a new type of treatment modality, nano-immunochemotherapy, for patients with cancer. In this review, we will discuss the development and potential applications of nanoscale platforms in medical diagnosis and treatment. To impact the care of patients with neurological diseases, advances in nanotechnology will require accelerated translation to the fields of brain mapping, CNS imaging, and nanoneurosurgery. Advances in nanoplatform, nano-imaging, and nano-drug delivery will drive the future development of nanomedicine, personalized medicine, and targeted therapy. We believe that the formation of a science, technology, medicine law–healthcare policy (STML) hub/center, which encourages collaboration among universities, medical centers, US government, industry, patient advocacy groups, charitable foundations, and philanthropists, could significantly facilitate such advancements and contribute to the translation of nanotechnology across medical disciplines.
PMCID: PMC3524337  PMID: 20149882
Nanoplatforms; Nanotechnology; Image-guided therapy; Nanomedicine; Nanoneurosurgery; Nanostructures; Contrast agents; Nanoparticles; Nanotechnology policy; Nano-radiology; Nano-neuroscience; Nano-neurology
12.  Poly(methyl malate) Nanoparticles: Formation, Degradation, and Encapsulation of Anticancer Drugs 
Macromolecular bioscience  2011;11(10):1370-1377.
PMLA nanoparticles with diameters of 150–250 nm are prepared, and their hydrolytic degradation is studied under physiological conditions. Degradation occurs by hydrolysis of the side chain methyl ester followed by cleavage of the main-chain ester group with methanol and L-malic acid as the final degradation products. No alteration of the cell viability is found after 1 h of incubation, but toxicity increases significantly after 3 d, probably due to the noxious effect of the released methanol. Anticancer drugs temozolomide and doxorubicin are encapsulated in the NPs with 20–40% efficiency, and their release is monitored using in vitro essays. Temozolomide is fully liberated within several hours, whereas doxorubicin is steadily released from the particles over a period of 1 month.
PMCID: PMC3496083  PMID: 21793213
anticancer DDS; biodegradable nanoparticles; polymalates; poly(malic acid)
13.  New functional degradable and bio-compatible nanoparticles based on poly(malic acid) derivatives for site-specific anti-cancer drug delivery 
Design of an efficient site-specific drug delivery system based on degradable functional polymers still remains challenging. In this work, we synthesized and characterized three degradable functional polyesters belonging to the poly(malic acid) family: the poly(benzyl malate) (PMLABe), the poly(ethylene glycol)-b-poly(benzyl malate) (PEG42-b-PMLABe), the biotin-poly(ethylene glycol)-bpoly( benzyl malate) (Biot-PEG62-PMLABe). Starting from these building blocks, we were able to prepare the corresponding well-defined degradable functional nanoparticles whose toxicity was evaluated in vitro on normal and cancer cell lines. Results have evidenced that the prepared nanoparticles did not show any significant cytotoxicity even at high concentrations. A model anti-cancer drug (doxorubicin, Dox) or a fluorescent probe (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, DiD oil) has been encapsulated into PMLABe, PEG42-PMLABe or Biot-PEG62-PMLABe based nanoparticles in order to evaluate, respectively, the in vitro cytotoxicity of Dox-loaded nanoparticles on normal and cancer cell lines and the ligand (biotin) effect on cellular uptake in vitro using mmt 060562 cell line. Dox-loaded PMLABe, PEG42-PMLABe or Biot-PEG62-PMLABe nanoparticles showed an in vitro cytotoxicity similar to that of free Dox. Moreover, the DiD oil loaded Biot-PEG62-PMLABe based nanoparticles showed a better in vitro cellular uptake than ligand-free DiD oil loaded nanoparticles. Both results evidence the great potential of such degradable functional poly(malic acid) derivatives for the design of highly efficient site-specific anti-cancer nanovectors.
PMCID: PMC3487703  PMID: 21550387
Degradable polyesters; Poly(malic acid) derivatives; Degradable functional nanoparticles; In vitro cytotoxicity; In vitro cellular uptake
14.  Inhibition of laminin-8 in vivo using a novel poly(malic acid)-based carrier reduces glioma angiogenesis 
Angiogenesis  2006;9(4):183-191.
We have previously shown that laminin-8, a vascular basement membrane component, was over-expressed in human glioblastomas multiforme and their adjacent tissues compared to normal brain. Increased laminin-8 correlated with shorter glioblastoma recurrence time and poor patient survival making it a potential marker for glioblastoma diagnostics and prediction of disease outcome. However, laminin-8 therapeutic potential was unknown because the technology of blocking the expression of multi-chain complex proteins was not yet developed. To inhibit the expression of laminin-8 constituents in glioblastoma in vitro and in vivo, we used Polycefin, a bioconjugate drug delivery system based on slime-mold Physarum polycephalum-derived poly(malic acid). It carries an attached transferrin receptor antibody to target tumor cells and to deliver two conjugated morpholino antisense oligonucleotides against laminin-8 α4 and β1 chains. Polycefin efficiently inhibited the expression of both laminin-8 chains by cultured glioblastoma cells. Intracranial Polycefin treatment of human U87MG glioblastoma-bearing nude rats reduced incorporation of both tumor-derived laminin-8 chains into vascular basement membranes. Polycefin was thus able to simultaneously inhibit the expression of two different chains of a complex protein. The treatment also significantly reduced tumor microvessel density (p < 0.001) and area (p < 0.001) and increased animal survival (p < 0.0004). These data suggest that laminin-8 may be important for glioblastoma angiogenesis. Polycefin, a versatile nanoscale drug delivery system, was suitable for in vivo delivery of two antisense oligonucleotides to brain tumor cells causing a reduction of glioblastoma angiogenesis and an increase of animal survival. This system may hold promise for future clinical applications.
PMCID: PMC3487708  PMID: 17109197
Tumor angiogenesis; Glioma; Laminin-8; Multiple drug targeting; Poly(malic acid)
15.  Polycefin, a New Prototype of a Multifunctional Nanoconjugate Based on Poly(β-l-malic acid) for Drug Delivery 
Bioconjugate chemistry  2006;17(2):317-326.
A new prototype of nanoconjugate, Polycefin, was synthesized for targeted delivery of antisense oligonucleotides and monoclonal antibodies to brain tumors. The macromolecular carrier contains: 1. biodegradable, nonimmunogenic, nontoxic β-poly(l-malic acid) of microbial origin; 2. Morpholino antisense oligonucleotides targeting laminin α4 and β1 chains of laminin-8, which is specifically overexpressed in glial brain tumors; 3. monoclonal anti-transferrin receptor antibody for specific tissue targeting; 4. oligonucleotide releasing disulfide units; 5. l-valine containing, pH-sensitive membrane disrupting unit(s), 6. protective poly(ethylene glycol); 7. a fluorescent dye (optional). Highly purified modules were conjugated directly with N-hydroxysuccinimidyl ester-activated β-poly-(l-malic acid) at pendant carboxyl groups or at thiol containing spacers via thioether and disulfide bonds. Products were chemically validated by physical, chemical, and functional tests. In vitro experiments using two human glioma cell lines U87MG and T98G demonstrated that Polycefin was delivered into the tumor cells by a receptor-mediated endocytosis mechanism and was able to inhibit the synthesis of laminin-8 α4 and β1 chains at the same time. Inhibition of laminin-8 expression was in agreement with the designed endosomal membrane disruption and drug releasing activity. In vivo imaging showed the accumulation of intravenously injected Polycefin in brain tumor tissue via the antibody-targeted transferrin receptor-mediated endosomal pathway in addition to a less efficient mechanism known for high molecular mass biopolymers as enhanced permeability and retention effect. Polycefin was nontoxic to normal and tumor astrocytes in a wide range of concentrations, accumulated in brain tumor, and could be used for specific targeting of several biomarkers simultaneously.
PMCID: PMC3487710  PMID: 16536461
16.  Nanoconjugate Platforms Development Based in Poly(β,L-Malic Acid) Methyl Esters for Tumor Drug Delivery 
Journal of nanotechnology  2010;2010:825363.
New copolyesters derived from poly(β,L-malic acid) have been designed to serve as nanoconjugate platforms in drug delivery. 25% and 50% methylated derivatives (coPMLA-Me25H75 and coPMLA-Me50H50) with absolute molecular weights of 32 600 Da and 33 100 Da, hydrodynamic diameters of 3.0 nm and 5.2 nm and zeta potential of −15mV and −8.25mV, respectively, were found to destabilize membranes of liposomes at pH 5.0 and pH 7.5 at concentrations above 0.05mg/mL. The copolymers were soluble in PBS (half life of 40 hours) and in human plasma (half life of 15 hours) but they showed tendency to aggregate at high levels of methylation. Fluorescence-labeled copolymers were internalized into MDA-MB-231 breast cancer cells with increased efficiency for the higher methylated copolymer. Viability of cultured brain and breast cancer cell lines indicated moderate toxicity that increased with methylation. The conclusion of the present work is that partially methylated poly(β,L-malic acid) copolyesters are suitable as nanoconjugate platforms for drug delivery.
PMCID: PMC3459346  PMID: 23024655
17.  Nanobiopolymer for Direct Targeting and Inhibition of EGFR Expression in Triple Negative Breast Cancer 
PLoS ONE  2012;7(2):e31070.
Treatment options for triple negative breast cancer (TNBC) are generally limited to cytotoxic chemotherapy. Recently, anti-epidermal growth factor receptor (EGFR) therapy has been introduced for TNBC patients. We engineered a novel nanobioconjugate based on a poly(β-L-malic acid) (PMLA) nanoplatform for TNBC treatment. The nanobioconjugate carries anti-tumor nucleosome-specific monoclonal antibody (mAb) 2C5 to target breast cancer cells, anti-mouse transferrin receptor (TfR) antibody for drug delivery through the host endothelial system, and Morpholino antisense oligonucleotide (AON) to inhibit EGFR synthesis. The nanobioconjugates variants were: (1) P (BioPolymer) with AON, 2C5 and anti-TfR for tumor endothelial and cancer cell targeting, and EGFR suppression (P/AON/2C5/TfR), and (2) P with AON and 2C5 (P/AON/2C5). Controls included (3) P with 2C5 but without AON (P/2C5), (4) PBS, and (5) P with PEG and leucine ester (LOEt) for endosomal escape (P/mPEG/LOEt). Drugs were injected intravenously to MDA-MB-468 TNBC bearing mice. Tissue accumulation of injected nanobioconjugates labeled with Alexa Fluor 680 was examined by Xenogen IVIS 200 (live imaging) and confocal microscopy of tissue sections. Levels of EGFR, phosphorylated and total Akt in tumor samples were detected by western blotting.
In vitro western blot showed that the leading nanobioconjugate P/AON/2C5/TfR inhibited EGFR synthesis significantly better than naked AON. In vivo imaging revealed that 2C5 increased drug-tumor accumulation. Significant tumor growth inhibition was observed in mice treated with the lead nanobioconjugate (1) [P = 0.03 vs. controls; P<0.05 vs. nanobioconjugate variant (2)]. Lead nanobioconjugate (1) also showed stronger inhibition of EGFR expression and Akt phosphorylation than other treatments. Treatment of TNBC with the new nanobioconjugate results in tumor growth arrest by inhibiting EGFR and its downstream signaling intermediate, phosphorylated Akt. The nanobioconjugate represents a new generation of nanodrugs for treatment of TNBC.
PMCID: PMC3280290  PMID: 22355336
18.  Cellular Delivery of Doxorubicin via pH-Controlled Hydrazone Linkage Using Multifunctional Nano Vehicle Based on Poly(β-L-Malic Acid) 
Doxorubicin (DOX) is currently used in cancer chemotherapy to treat many tumors and shows improved delivery, reduced toxicity and higher treatment efficacy when being part of nanoscale delivery systems. However, a major drawback remains its toxicity to healthy tissue and the development of multi-drug resistance during prolonged treatment. This is why in our work we aimed to improve DOX delivery and reduce the toxicity by chemical conjugation with a new nanoplatform based on polymalic acid. For delivery into recipient cancer cells, DOX was conjugated via pH-sensitive hydrazone linkage along with polyethylene glycol (PEG) to a biodegradable, non-toxic and non-immunogenic nanoconjugate platform: poly(β-l-malic acid) (PMLA). DOX-nanoconjugates were found stable under physiological conditions and shown to successfully inhibit in vitro cancer cell growth of several invasive breast carcinoma cell lines such as MDA-MB-231 and MDA-MB- 468 and of primary glioma cell lines such as U87MG and U251.
PMCID: PMC3472769  PMID: 23109877
polymalic acid; doxorubicin; nanoconjugate; pH-controlled hydrazine linkage; brain and breast cancer
19.  Temozolomide Delivery to Tumor Cells by a Multifunctional Nano Vehicle Based on Poly(β-L-malic acid) 
Pharmaceutical research  2010;27(11):2317-2329.
Temozolomide (TMZ) is a pro-drug releasing a DNA alkylating agent that is the most effective drug to treat glial tumors when combined with radiation. TMZ is toxic, and therapeutic dosages are limited by severe side effects. Targeted delivery is thus needed to improve efficiency and reduce non-tumor tissue toxicity.
Multifunctional targetable nanoconjugates of TMZ hydrazide were synthesized using poly(β-L-malic acid) platform, which contained a targeting monoclonal antibody to transferrin receptor (TfR), trileucine (LLL), for pH-dependent endosomal membrane disruption, and PEG for protection.
The water-soluble TMZ nanoconjugates had hydrodynamic diameters in the range of 6.5 to 14.8 nm and ζ potentials in the range of −6.3 to −17.7 mV. Fifty percent degradation in human plasma was observed in 40 h at 37°C. TMZ conjugated with polymer had a half-life of 5–7 h, compared with 1.8 h for free TMZ. The strongest reduction of human brain and breast cancer cell viability was obtained by versions of TMZ nanoconjugates containing LLL and anti-TfR antibody. TMZ-resistant cancer cell lines were sensitive to TMZ nanoconjugate treatment.
TMZ-polymer nanoconjugates entered the tumor cells by receptor-mediated endocytosis, effectively reduced cancer cell viability, and can potentially be used for targeted tumor treatment.
PMCID: PMC2952070  PMID: 20387095
anti-TfR mAb; nanoconjugate; pH-dependent membrane disruption; polymalic acid; targeted drug delivery; temozolomide
Brain research bulletin  2009;81(2-3):262.
Our previous data suggested the involvement of matrix metalloproteinase-10 (MMP-10) and cathepsin F (CTSF) in the basement membrane and integrin changes occurring in diabetic corneas. These markers were now examined in normal human organ-cultured corneas upon recombinant adenovirus (rAV)-driven transduction of MMP-10 and CTSF genes.
Fifteen pairs of normal autopsy human corneas were used. One cornea of each pair was transduced with rAV expressing either CTSF or MMP-10 genes. 1–2 × 108 plaque forming units of rAV per cornea were added to cultures for 48 hr with or without sildenafil citrate. The fellow cornea of each pair received control rAV with vector alone. After 6–10 days incubation without rAV, corneas were analyzed by Western blot or immunohistochemistry, or tested for healing of 5-mm circular epithelial wounds caused by topical application of n-heptanol.
Sildenafil significantly increased epithelial transduction efficiency, apparently through stimulation of rAV endocytosis through caveolae. Corneas transduced with CTSF or MMP-10 genes or their combination had increased epithelial immunostaining of respective proteins compared to fellow control corneas. Staining for diabetic markers integrin α3β1, nidogen-1, nidogen-2, and laminin γ2 chain became weaker and irregular upon proteinase transduction. Expression of phosphorylated Akt was decreased in proteinase-transduced corneas. Joint overexpression of both proteinases led to significantly slower corneal wound healing that became similar to that observed in diabetic ones.
The data suggest that MMP-10 and CTSF may be responsible for abnormal marker patterns and impaired wound healing in diabetic corneas. Inhibition of these proteinases in diabetic corneas may alleviate diabetic keratopathy symptoms.
PMCID: PMC2815249  PMID: 19828126
diabetic cornea; organ culture; MMP-10; cathepsin F; Akt; sildenafil
21.  Phosphodiesterase Type 5 Inhibitors Increase Herceptin Transport and Treatment Efficacy in Mouse Metastatic Brain Tumor Models 
PLoS ONE  2010;5(4):e10108.
Chemotherapeutic drugs and newly developed therapeutic monoclonal antibodies are adequately delivered to most solid and systemic tumors. However, drug delivery into primary brain tumors and metastases is impeded by the blood-brain tumor barrier (BTB), significantly limiting drug use in brain cancer treatment.
Methodology/Principal Findings
We examined the effect of phosphodiesterase 5 (PDE5) inhibitors in nude mice on drug delivery to intracranially implanted human lung and breast tumors as the most common primary tumors forming brain metastases, and studied underlying mechanisms of drug transport. In vitro assays demonstrated that PDE5 inhibitors enhanced the uptake of [14C]dextran and trastuzumab (Herceptin®, a humanized monoclonal antibody against HER2/neu) by cultured mouse brain endothelial cells (MBEC). The mechanism of drug delivery was examined using inhibitors for caveolae-mediated endocytosis, macropinocytosis and coated pit/clathrin endocytosis. Inhibitor analysis strongly implicated caveolae and macropinocytosis endocytic pathways involvement in the PDE5 inhibitor-enhanced Herceptin uptake by MBEC. Oral administration of PDE5 inhibitor, vardenafil, to mice with HER2-positive intracranial lung tumors led to an increased tumor permeability to high molecular weight [14C]dextran (2.6-fold increase) and to Herceptin (2-fold increase). Survival time of intracranial lung cancer-bearing mice treated with Herceptin in combination with vardenafil was significantly increased as compared to the untreated, vardenafil- or Herceptin-treated mice (p<0.01). Log-rank survival analysis of mice bearing HER2-positive intracranial breast tumor also showed a significant survival increase (p<0.02) in the group treated with Herceptin plus vardenafil as compared to other groups. However, vardenafil did not exert any beneficial effect on survival of mice bearing intracranial breast tumor with low HER2 expression and co-treated with Herceptin (p>0.05).
These findings suggest that PDE5 inhibitors may effectively modulate BTB permeability, and enhance delivery and therapeutic efficacy of monoclonal antibodies in hard-to-treat brain metastases from different primary tumors that had metastasized to the brain.
PMCID: PMC2856671  PMID: 20419092
22.  Poly(malic acid) nanoconjugates containing various antibodies and oligonucleotides for multitargeting drug delivery 
Nanomedicine (London, England)  2008;3(2):247-265.
Nanoconjugates are emerging as promising drug-delivery vehicles because of their multimodular structure enabling them to actively target discrete cells, pass through biological barriers and simultaneously carry multiple drugs of various chemical nature. Nanoconjugates have matured from simple devices to multifunctional, biodegradable, nontoxic and nonimmunogenic constructs, capable of delivering synergistically functioning drugs in vivo. This review mainly concerns the Polycefin family of natural-derived polymeric drug-delivery devices as an example. This type of vehicle is built by hierarchic conjugation of functional groups onto the backbone of poly(malic acid), an aliphatic polyester obtained from the microorganism Physarum polycephalum. Particular Polycefin variants target human brain and breast tumors implanted into animals specifically and actively and could be detected easily by noninvasive imaging analysis. Delivery of antisense oligonucleotides to a tumor-specific angiogenic marker using Polycefin resulted in significant inhibition of tumor angiogenesis and increase of animal survival.
PMCID: PMC2734385  PMID: 18373429
biodegradable; brain cancer; breast cancer; imaging analysis; multiple antibodies; multiple drug delivery; multitargeting; Polycefin; poly(malic acid); tumor angiogenesis
23.  Nanoconjugate based on polymalic acid for tumor targeting 
Chemico-biological interactions  2007;171(2):195-203.
A new prototype of polymer-derived drug delivery system, the nanoconjugate Polycefin, was tested for its ability to accumulate in tumors based on enhanced permeability and retention (EPR) effect and receptor mediated endocytosis. Polycefin was synthesized for targeted delivery of Morpholino antisense oligonucleotides into certain tumors. It consists of units that are covalently conjugated with poly(β-L-malic acid) (Mw 50,000, Mw/Mn 1.3) highly purified from cultures of myxomycete Physarum polycephalum. The units are active in endosomal uptake, disruption of endosomal membranes, oligonucleotide release in the cytoplasm, and protection against enzymatic degradation in the vascular system. The polymer is biodegradable, non-immunogenic and non-toxic. Polycefin was also coupled with AlexaFluor 680 C2-maleimide dye for in vivo detection.
Nude mice received subcutaneous injections of MDA-MB 468 human breast cancer cells into the left posterior mid-dorsum or intracranial injections of human glioma cell line U87MG. Polycefin at concentration of 2.5 mg/kg was injected via the tail vein. In vivo fluorescence tumor imaging was performed at different time points, 0–180 min up to 24 h after the drug injection. The custom-made macro-illumination imaging MISTI system was used to examine the in vivo drug accumulation in animals bearing human breast and brain tumors. In breast tumors the fluorescence signal in large blood vessels and in the tumor increased rapidly until 60 min and remained in the tumor at a level 6 times higher than in non-tumor tissue (180 min) (p < 0.003). In brain tumors drug accumulated selectively in 24 h without any detectable signal in non-tumor areas. The results of live imaging were corroborated histologically by fluorescence microscopic examination of various organs. In addition to tumors, only kidney and liver showed some fluorescent signal. © 2007 Elsevier Ireland Ltd. All rights reserved.
PMCID: PMC2329596  PMID: 17376417
Brain glioma; Breast cancer; EPR effect; Fluorescence imaging; Drug delivery system; Poly(malic acid)
24.  Brain tumor tandem targeting using a combination of monoclonal antibodies attached to biopoly(β-L-malic acid) 
Tumor-specific targeting using achievements of nanotechnology is a mainstay of increasing efficacy of anti-tumor drugs. To improve drug targeting we covalently conjugated for the first time two different monoclonal antibodies, an anti-mouse transferrin receptor antibody and a mouse autoimmune anti-nucleosome antibody 2C5, onto the drug delivery nanoplatform, poly(β-L-malic acid). The active anti-tumor drug components attached to the same carrier molecule were antisense oligonucleotides to vascular protein laminin-8. The resulting drug, a new Polycefin variant, was administered intravenously into glioma-bearing xenogeneic animals. The drug delivery system was targeted across mouse endothelial system by the anti-mouse transferring receptor antibody and to the tumor cell surface by the anti-nucleosome antibody 2C5. The targeting efficacies of the Polycefin variants bearing either two antibodies or each single antibody were compared in vitro and in vivo. ELISA confirmed the co-existence of two antibodies on the same nanoplatform molecule and their functional activities. Fluorescence imaging analysis after 24 h of intravenous injection demonstrated significantly higher tumor accumulation of Polycefin variants with the tandem configuration of antibodies than with single antibodies. The results suggest improved efficacy for tandem configuration of antibodies than for single configurations carried by a drug delivery vehicle.
PMCID: PMC2394675  PMID: 17630012
Enhanced tumor targeting; Antibody tandem configuration; Nanobiopolymer; Brain tumor; Poly(β-L-malic acid)
25.  Proteinase and Growth Factor Alterations Revealed by Gene Microarray Analysis of Human Diabetic Corneas 
PURPOSE. To identify proteinases and growth factors abnormally expressed in human corneas of donors with diabetic retinopathy (DR), additional to previously described matrix metalloproteinase (MMP)-10 and -3 and insulin-like growth factor (IGF)-I.
METHODS. RNA was isolated from 35 normal, diabetic, and DR autopsy human corneas ex vivo or after organ culture. Amplified cRNA was analyzed using 22,000-gene microarrays (Agi-lent Technologies, Palo Alto, CA). Gene expression in each diabetic corneal cRNA was assessed against pooled cRNA from 7 to 9 normal corneas. Select differentially expressed genes were validated by quantitative real-time RT-PCR (QPCR) and immunohistochemistry. Organ cultures were treated with a cathepsin inhibitor, cystatin C, or MMP-10.
RESULTS. More than 100 genes were upregulated and 2200 were downregulated in DR corneas. Expression of cathepsin F and hepatocyte growth factor (HGF) genes was increased in ex vivo and organ-cultured DR corneas compared with normal corneas. HGF receptor c-met, fibroblast growth factor (FGF)-3, its receptor FGFR3, tissue inhibitor of metalloproteinase (TIMP)-4, laminin α4 chain, and thymosin β4 genes were down-regulated. The data were corroborated by QPCR and immuno-histochemistry analyses; main changes of these components occurred in corneal epithelium. In organ-cultured DR corneas, cystatin C increased laminin-10 and integrin α3β1, whereas in normal corneas MMP-10 decreased laminin-10 and integrin α3β1 expression.
CONCLUSIONS. Elevated cathepsin F and the ability of its inhibitor to produce a more normal phenotype in diabetic corneas suggest increased proteolysis in these corneas. Proteinase changes may result from abnormalities of growth factors, such as HGF and FGF-3, in DR corneas. Specific modulation of proteinases and growth factors could reduce diabetic corneal epitheliopathy.
PMCID: PMC1459105  PMID: 16186340

Results 1-25 (26)