In JoVE (1)

Other Publications (31)

Articles by Julia Y. Ljubimova in JoVE

Other articles by Julia Y. Ljubimova on PubMed

Antisense Inhibition of Laminin-8 Expression Reduces Invasion of Human Gliomas in Vitro

Molecular Cancer Therapeutics. Oct, 2003  |  Pubmed ID: 14578463

Using gene array technology, we recently observed for the first time an up-regulation of laminin alpha4 chain in human gliomas. The data were validated by semiquantitative reverse transcription-PCR for RNA expression and immunohistochemistry for protein expression. Moreover, increase of the alpha4 chain-containing laminin-8 correlated with poor prognosis for patients with brain gliomas. Therefore, we hypothesized that inhibition of laminin-8 expression by a new generation of highly specific and stable antisense oligonucleotides (Morpholino) against chains of laminin-8 could slow or stop the spread of glioma and its recurrence and thus might be a promising approach for glioma therapy. We next sought to establish an in vitro model to test the feasibility of this approach and to optimize conditions for Morpholino treatment. To develop a model, we used human glioblastoma multiforme cell lines M059K and U-87MG cocultured with normal human brain microvascular endothelial cells (HBMVEC). Using Western blot analysis and immunohistochemistry, we confirmed that antisense treatment effectively blocked laminin-8 protein synthesis. Antisense oligonucleotides against both alpha4 and beta1 chains of laminin-8 were able to block significantly the invasion of cocultures through Matrigel. On average, the invasion was blocked by 62% in cocultures of U-87MG with HBMVEC and by 53% in cocultures of M059K with HBMVEC. The results show that laminin-8 may contribute to glioma progression and recurrence not only as part of the neovascularization process but also by directly increasing the invasive potential of tumor cells.

Association Between Laminin-8 and Glial Tumor Grade, Recurrence, and Patient Survival

Cancer. Aug, 2004  |  Pubmed ID: 15274074

The authors previously sought to identify novel markers of glioma invasion and recurrence. Their research demonstrated that brain gliomas overexpressed a subset of vascular basement components, laminins, that contained the alpha4 chain. One of these laminins, laminin-8, was found to be present in highly invasive and malignant glioblastoma multiforme (GBM) (Grade 4 astrocytoma); its expression was associated with a decreased time to tumor recurrence, and it was found in vitro to promote invasion of GBM cell lines.

Overexpression of Beta1-chain-containing Laminins in Capillary Basement Membranes of Human Breast Cancer and Its Metastases

Breast Cancer Research : BCR. 2005  |  Pubmed ID: 15987446

Laminins are the major components of vascular and parenchymal basement membranes. We previously documented a switch in the expression of vascular laminins containing the alpha4 chain from predominantly laminin-9 (alpha4beta2gamma1) to predominantly laminin-8 (alpha4beta1gamma1) during progression of human brain gliomas to high-grade glioblastoma multiforme. Here, differential expression of laminins was studied in blood vessels and ductal epithelium of the breast.

Proteinase and Growth Factor Alterations Revealed by Gene Microarray Analysis of Human Diabetic Corneas

Investigative Ophthalmology & Visual Science. Oct, 2005  |  Pubmed ID: 16186340

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.

Changes in Laminin Isoforms Associated with Brain Tumor Invasion and Angiogenesis

Frontiers in Bioscience : a Journal and Virtual Library. 2006  |  Pubmed ID: 16146715

Laminins are the major constituents of blood vessel basement membranes (BMs). Each laminin is a trimer consisting of three assembled polypeptide chains, alpha, beta and gamma. 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 (alpha4beta2gamma1) is switched to laminin-8 (alpha4beta1gamma1), 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.

Polycefin, a New Prototype of a Multifunctional Nanoconjugate Based on Poly(beta-L-malic Acid) for Drug Delivery

Bioconjugate Chemistry. Mar-Apr, 2006  |  Pubmed ID: 16536461

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 beta-poly(L-malic acid) of microbial origin; 2. Morpholino antisense oligonucleotides targeting laminin alpha4 and beta1 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 beta-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 alpha4 and beta1 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.

Inhibition of Laminin-8 in Vivo Using a Novel Poly(malic Acid)-based Carrier Reduces Glioma Angiogenesis

Angiogenesis. 2006  |  Pubmed ID: 17109197

We have previously shown that laminin-8, a vascular basement membrane component, was overexpressed 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 alpha4 and beta1 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.

Brain Tumor Tandem Targeting Using a Combination of Monoclonal Antibodies Attached to Biopoly(beta-L-malic Acid)

Journal of Controlled Release : Official Journal of the Controlled Release Society. Oct, 2007  |  Pubmed ID: 17630012

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(beta-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.

Nanoconjugate Based on Polymalic Acid for Tumor Targeting

Chemico-biological Interactions. Jan, 2008  |  Pubmed ID: 17376417

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(beta-l-malic acid) (M(w) 50,000, M(w)/M(n) 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 2.5mg/kg was injected via the tail vein. In vivo fluorescence tumor imaging was performed at different time points, 0-180 min up to 24h 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 24h 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.

Poly(malic Acid) Nanoconjugates Containing Various Antibodies and Oligonucleotides for Multitargeting Drug Delivery

Nanomedicine (London, England). Apr, 2008  |  Pubmed ID: 18373429

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.

Adenovirus-driven Overexpression of Proteinases in Organ-cultured Normal Human Corneas Leads to Diabetic-like Changes

Brain Research Bulletin. Feb, 2010  |  Pubmed ID: 19828126

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 x 10(8) plaque forming units of rAV per cornea were added to cultures for 48 h with or without sildenafil citrate. The fellow cornea of each pair received control rAV with vector alone. After 6-10 days additional 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 by 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 alpha(3)beta(1), nidogen-1, nidogen-2, and laminin gamma2 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 corneas. 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.

Temozolomide Delivery to Tumor Cells by a Multifunctional Nano Vehicle Based on Poly(β-L-malic Acid)

Pharmaceutical Research. Nov, 2010  |  Pubmed ID: 20387095

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.

Phosphodiesterase Type 5 Inhibitors Increase Herceptin Transport and Treatment Efficacy in Mouse Metastatic Brain Tumor Models

PloS One. 2010  |  Pubmed ID: 20419092

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.

Inhibition of Brain Tumor Growth by Intravenous Poly (β-L-malic Acid) Nanobioconjugate with PH-dependent Drug Release [corrected]

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2010  |  Pubmed ID: 20921419

Effective treatment of brain neurological disorders such as Alzheimer's disease, multiple sclerosis, or tumors should be possible with drug delivery through blood-brain barrier (BBB) or blood-brain tumor barrier (BTB) and targeting specific types of brain cells with drug release into the cell cytoplasm. A polymeric nanobioconjugate drug based on biodegradable, nontoxic, and nonimmunogenic polymalic acid as a universal delivery nanoplatform was used for design and synthesis of nanomedicine drug for i.v. treatment of brain tumors. The polymeric drug passes through the BTB and tumor cell membrane using tandem monoclonal antibodies targeting the BTB and tumor cells. The next step for polymeric drug action was inhibition of tumor angiogenesis by specifically blocking the synthesis of a tumor neovascular trimer protein, laminin-411, by attached antisense oligonucleotides (AONs). The AONs were released into the target cell cytoplasm via pH-activated trileucine, an endosomal escape moiety. Drug delivery to the brain tumor and the release mechanism were both studied for this nanobiopolymer. Introduction of a trileucine endosome escape unit resulted in significantly increased AON delivery to tumor cells, inhibition of laminin-411 synthesis in vitro and in vivo, specific accumulation in brain tumors, and suppression of intracranial glioma growth compared with pH-independent leucine ester. The availability of a systemically active polymeric drug delivery system that passes through the BTB, targets tumor cells, and inhibits glioma growth gives hope for a successful strategy of glioma treatment. This delivery system with drug release into the brain-specific cell type could be useful for treatment of various brain pathologies.

Nanoconjugate Platforms Development Based in Poly(β,L-Malic Acid) Methyl Esters for Tumor Drug Delivery

Journal of Nanotechnology. 2010  |  Pubmed ID: 23024655

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-Me(25)H(75) and coPMLA-Me(50)H(50)) 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.

Nanoplatforms for Constructing New Approaches to Cancer Treatment, Imaging, and Drug Delivery: What Should Be the Policy?

NeuroImage. Jan, 2011  |  Pubmed ID: 20149882

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.

Polymalic Acid-based Nanobiopolymer Provides Efficient Systemic Breast Cancer Treatment by Inhibiting Both HER2/neu Receptor Synthesis and Activity

Cancer Research. Feb, 2011  |  Pubmed ID: 21303974

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.

The Optimization of Polymalic Acid Peptide Copolymers for Endosomolytic Drug Delivery

Biomaterials. Aug, 2011  |  Pubmed ID: 21514661

Membranolytic macromolecules are promising vehicles for cytoplasmic 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 pendent 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 tripeptides of leucine, tryptophan and phenylalanine were pH-dependent in contrast to pH-independent copolymers of Leucine ethyl ester and Leu-Leu-Leu-NH(2) 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.

Poly(methyl Malate) Nanoparticles: Formation, Degradation, and Encapsulation of Anticancer Drugs

Macromolecular Bioscience. Oct, 2011  |  Pubmed ID: 21793213

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.

New Functional Degradable and Bio-compatible Nanoparticles Based on Poly(malic Acid) Derivatives for Site-specific Anti-cancer Drug Delivery

International Journal of Pharmaceutics. Feb, 2012  |  Pubmed ID: 21550387

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) (PEG(42)-b-PMLABe), the biotin-poly(ethylene glycol)-b-poly(benzyl malate) (Biot-PEG(62)-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, PEG(42)-PMLABe or Biot-PEG(62)-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, PEG(42)-PMLABe or Biot-PEG(62)-PMLABe nanoparticles showed an in vitro cytotoxicity similar to that of free Dox. Moreover, the DiD oil loaded Biot-PEG(62)-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.

The Transferrin Receptor and the Targeted Delivery of Therapeutic Agents Against Cancer

Biochimica Et Biophysica Acta. Mar, 2012  |  Pubmed ID: 21851850

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.

Nanobiopolymer for Direct Targeting and Inhibition of EGFR Expression in Triple Negative Breast Cancer

PloS One. 2012  |  Pubmed ID: 22355336

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.

Cellular Delivery of Doxorubicin Via PH-Controlled Hydrazone Linkage Using Multifunctional Nano Vehicle Based on Poly(β-L-Malic Acid)

International Journal of Molecular Sciences. 2012  |  Pubmed ID: 23109877

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.

Biocompatible Nanopolymers: the Next Generation of Breast Cancer Treatment?

Nanomedicine (London, England). Oct, 2012  |  Pubmed ID: 23148535

Distinct Mechanisms of Membrane Permeation Induced by Two Polymalic Acid Copolymers

Biomaterials. Jan, 2013  |  Pubmed ID: 23063368

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.

Gene Expression Changes in Rat Brain After Short and Long Exposures to Particulate Matter in Los Angeles Basin Air: Comparison with Human Brain Tumors

Experimental and Toxicologic Pathology : Official Journal of the Gesellschaft Für Toxikologische Pathologie. Nov, 2013  |  Pubmed ID: 23688656

Air pollution negatively impacts pulmonary, cardiovascular, and central nervous systems. Although its influence on brain cancer is unclear, toxic pollutants can cause blood-brain barrier disruption, enabling them to reach the brain and cause alterations leading to tumor development. By gene microarray analysis validated by quantitative RT-PCR and immunostaining we examined whether rat (n=104) inhalation exposure to air pollution particulate matter (PM) resulted in brain molecular changes similar to those associated with human brain tumors. Global brain gene expression was analyzed after exposure to PM (coarse, 2.5-10μm; fine, <2.5μm; or ultrafine, <0.15μm) and purified air for different times, short (0.5, 1, and 3 months) and chronic (10 months), for 5h per day, four days per week. Expression of select gene products was also studied in human brain (n=7) and in tumors (n=83). Arc/Arg3.1 and Rac1 genes, and their protein products were selected for further examination. Arc was elevated upon two-week to three-month exposure to coarse PM and declined after 10-month exposure. Rac1 was significantly elevated upon 10-month coarse PM exposure. On human brain tumor sections, Arc was expressed in benign meningiomas and low-grade gliomas but was much lower in high-grade tumors. Conversely, Rac1 was elevated in high-grade vs. low-grade gliomas. Arc is thus associated with early brain changes and low-grade tumors, whereas Rac1 is associated with long-term PM exposure and highly aggressive tumors. In summary, exposure to air PM leads to distinct changes in rodent brain gene expression similar to those observed in human brain tumors.

Polymalic Acid Nanobioconjugate for Simultaneous Immunostimulation and Inhibition of Tumor Growth in HER2/neu-positive Breast Cancer

Journal of Controlled Release : Official Journal of the Controlled Release Society. Nov, 2013  |  Pubmed ID: 23770212

Breast cancer remains the second leading cause of cancer death among women in the United States. Breast cancer prognosis is particularly poor in case of tumors overexpressing the oncoprotein HER2/neu. A new nanobioconjugate of the Polycefin(TM) 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 ensure 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 retained the biological activity of IL-2. We also showed the uptake of the nanobioconjugate into HER2/neu-positive breast cancer cells and enhanced tumor targeting in vivo. The nanobioconjugate exhibited marked anti-tumor activity manifested by significantly longer animal survival and significantly increased anti-HER2/neu immune response in immunocompetent mice bearing D2F2/E2 murine mammary tumors that express human HER2/neu. The combination of laminin-411 AON and antibody-cytokine fusion protein on a single polymeric platform results in a new nanobioconjugate that can act against cancer cells through inhibition of tumor growth and angiogenesis and the orchestration of an immune response against the tumor. The present Polycefin(TM) variant may be a promising agent for treating HER2/neu expressing tumors and demonstrates the versatility of the Polycefin(TM) nanobioconjugate platform.

Toxicity and Efficacy Evaluation of Multiple Targeted Polymalic Acid Conjugates for Triple-negative Breast Cancer Treatment

Journal of Drug Targeting. Dec, 2013  |  Pubmed ID: 24032759

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 nanoconjugate 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.

Nanomedicine Therapeutic Approaches to Overcome Cancer Drug Resistance

Advanced Drug Delivery Reviews. Nov, 2013  |  Pubmed ID: 24120656

Nanomedicine is an emerging form of therapy that focuses on alternative drug delivery and improvement of the treatment efficacy while reducing detrimental side effects to normal tissues. Cancer drug resistance is a complicated process that involves multiple mechanisms. Here we discuss the major forms of drug resistance and the new possibilities that nanomedicines offer to overcome these treatment obstacles. Novel nanomedicines that have a high ability for flexible, fast drug design and production based on tumor genetic profiles can be created making drug selection for personal patient treatment much more intensive and effective. This review aims to demonstrate the advantage of the young medical science field, nanomedicine, for overcoming cancer drug resistance. With the advanced design and alternative mechanisms of drug delivery known for different nanodrugs including liposomes, polymer conjugates, micelles, dendrimers, carbon-based, and metallic nanoparticles, overcoming various forms of multi-drug resistance looks promising and opens new horizons for cancer treatment.

Near-infrared Imaging of Brain Tumors Using the Tumor Paint BLZ-100 to Achieve Near-complete Resection of Brain Tumors

Neurosurgical Focus. Feb, 2014  |  Pubmed ID: 24484247

The intraoperative clear delineation between brain tumor and normal tissue in real time is required to ensure near-complete resection without damaging the nearby eloquent brain. Tumor Paint BLZ-100, a tumor ligand chlorotoxin (CTX) conjugated to indocyanine green (ICG), has shown potential to be a targeted contrast agent. There are many infrared imaging systems in use, but they are not optimized to the low concentration and amount of ICG. The authors present a novel proof-of-concept near-infrared (NIR) imaging system using a standard charge-coupled device (CCD) camera for visualizing low levels of ICG attached to the tumors. This system is small, inexpensive, and sensitive. The imaging system uses a narrow-band laser at 785 nm and a notch filter in front of the sensor at the band. The camera is a 2-CCD camera, which uses identical CCDs for both visible and NIR light.

Multilayer Films Assembled from Naturally-derived Materials for Controlled Protein Release

Biomacromolecules. Jun, 2014  |  Pubmed ID: 24825478

Herein we designed and characterized films composed of naturally derived materials for controlled release of proteins. Traditional drug delivery strategies rely on synthetic or semisynthetic materials or utilize potentially denaturing assembly conditions that are not optimal for sensitive biologics. Layer-by-layer (LbL) assembly of films uses benign conditions and can generate films with various release mechanisms including hydrolysis-facilitated degradation. These use components such as synthetic polycations that degrade into non-natural products. Herein we report the use of a naturally derived, biocompatible and degradable polyanion, poly(β-l-malic acid), alone and in combination with chitosan in an LbL film, whose degradation products of malic acid and chitosan are both generally recognized as safe (GRAS) by the FDA. We have found that films based on this polyanion have shown sustained release of a model protein, lysozyme that can be timed from tens of minutes to multiple days through different film architectures. We also report the incorporation and release of a clinically used biologic, basic fibroblast growth factor (bFGF), which demonstrates the use of this strategy as a platform for controlled release of various biologics.

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