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1.  Biocompatible nanopolymers: the next generation of breast cancer treatment? 
Nanomedicine (London, England)  2012;7(10):1467-1470.
doi:10.2217/nnm.12.115
PMCID: PMC4086916  PMID: 23148535
biomarkers; breast cancer; nanodrugs; nanopolymers; personalized medicine; polymalic acid
2.  Curcumin Targeted, Polymalic Acid-Based MRI Contrast Agent for the Detection of Aβ Plaques in Alzheimer’s Diseasea 
Macromolecular bioscience  2015;15(9):1212-1217.
Currently, there is no gadolinium-based contrast agent available for conventional magnetic resonance imaging (MRI) detection of amyloidal beta (Aβ) plaques in Alzheimer’s disease (AD). Its timely finding would be vital for patient survival and quality of life. Curcumin (CUR), a common Indian spice effectively binds to Aβ plaques which is a hallmark of AD. To address this binding, we have designed a novel nanoimaging agent (NIA) based on nature-derived poly(β-l-malic acid) (PMLA) containing covalently attached gadolinium–DOTA(Gd–DOTA) and nature-derived CUR. The all-in-one agent recognizes and selectively binds to Aβ plaques and is detected by MRI. It efficiently detected Aβ plaques in human and mouse samples by an ex vivo staining. The method can be useful in clinic for safe and noninvasive diagnosis of AD.
Graphical abstract
doi:10.1002/mabi.201500062
PMCID: PMC4794283  PMID: 26036700
Alzheimer disease; curcumin; MRI; nanoimaging agent; polymalic acid
3.  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.
doi:10.3109/1061186X.2013.837470
PMCID: PMC4043297  PMID: 24032759
Hematologic; immunogenicity; in vivo treatment; nanoconjugate drugs; polymalic acid; toxicity; triple-negative breast cancer
4.  MRI Virtual Biopsy and Treatment of Brain Metastatic Tumors with Targeted Nanobioconjugates: Nanoclinic in the Brain 
ACS nano  2015;9(5):5594-5608.
Differential diagnosis of brain magnetic resonance imaging (MRI) enhancement(s) remains a significant problem, which may be difficult to resolve without biopsy, which can be often dangerous or even impossible. Such MRI enhancement(s) can result from metastasis of primary tumors such as lung or breast, radiation necrosis, infections, or a new primary brain tumor (glioma, meningioma). Neurological symptoms are often the same on initial presentation. To develop a more precise noninvasive MRI diagnostic method, we have engineered a new class of poly(β-L-malic acid) polymeric nanoimaging agents (NIAs). The NIAs carrying attached MRI tracer are able to pass through the blood–brain barrier (BBB) and specifically target cancer cells for efficient imaging. A qualitative/quantitative “MRI virtual biopsy” method is based on a nanoconjugate carrying MRI contrast agent gadolinium-DOTA and antibodies recognizing tumor-specific markers and extravasating through the BBB. In newly developed double tumor xenogeneic mouse models of brain metastasis this noninvasive method allowed differential diagnosis of HER2- and EGFR-expressing brain tumors. After MRI diagnosis, breast and lung cancer brain metastases were successfully treated with similar tumor-targeted nanoconjugates carrying molecular inhibitors of EGFR or HER2 instead of imaging contrast agent. The treatment resulted in a significant increase in animal survival and markedly reduced immunostaining for several cancer stem cell markers. Novel NIAs could be useful for brain diagnostic MRI in the clinic without currently performed brain biopsies. This technology shows promise for differential MRI diagnosis and treatment of brain metastases and other pathologies when biopsies are difficult to perform.
Graphical abstract
doi:10.1021/acsnano.5b01872
PMCID: PMC4768903  PMID: 25906400
nanoconjugate; brain metastasis; MRI; nanomedicine; blood; brain barrier; tumor treatment; tumor targeting
5.  CHANGES IN LAMININ ISOFORMS ASSOCIATED WITH BRAIN TUMOR INVASION AND ANGIOGENESIS 
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
6.  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.
doi:10.1158/0008-5472.CAN-10-3093
PMCID: PMC3428373  PMID: 21303974
7.  Ordered and Kinetically Discrete Sequential Protein Release from Biodegradable Thin Films** 
Multidrug regimens can sometimes treat recalcitrant diseases when single-drug therapies fail. Recapitulating complex multidrug administration from controlled release films for localized delivery remains challenging because their release kinetics are frequently intertwined and an initial burst release of each drug is usually uncontrollable. Herein we demonstrate kinetic control over protein release by crosslinking Layer-by-Layer films during the assembly process. We used biodegradable and naturally derived components and relied on copper-free click chemistry for bioorthogonal covalent crosslinks throughout the film that entrap, but do not modify the embedded protein. We found that this strategy restricted the interdiffusion of protein while maintaining its activity. By depositing a barrier layer and a second protein-containing layer atop this construct, we generated well-defined sequential protein release with minimal overlap that follows their spatial distribution within the film.
doi:10.1002/anie.201403702
PMCID: PMC4387866  PMID: 24938739
staged release; drug delivery; polyelectrolyte multilayers; controlled release; click chemistry
8.  Multilayer Films Assembled from Naturally-Derived Materials for Controlled Protein Release 
Biomacromolecules  2014;15(6):2049-2057.
Herein we designed and characterized films composed of naturally derived materials for controlled release of proteins. Traditional drug delivery strategies rely on synthetic or semi-synthetic 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.
doi:10.1021/bm5001839
PMCID: PMC4387870  PMID: 24825478
Drug delivery; layer-by-layer; growth factor; sustained release
9.  Quantitative Analysis of PMLA Nanoconjugate Components after Backbone Cleavage 
Multifunctional polymer nanoconjugates containing multiple components show great promise in cancer therapy, but in most cases complete analysis of each component is difficult. Polymalic acid (PMLA) based nanoconjugates have demonstrated successful brain and breast cancer treatment. They consist of multiple components including targeting antibodies, Morpholino antisense oligonucleotides (AONs), and endosome escape moieties. The component analysis of PMLA nanoconjugates is extremely difficult using conventional spectrometry and HPLC method. Taking advantage of the nature of polyester of PMLA, which can be cleaved by ammonium hydroxide, we describe a method to analyze the content of antibody and AON within nanoconjugates simultaneously using SEC-HPLC by selectively cleaving the PMLA backbone. The selected cleavage conditions only degrade PMLA without affecting the integrity and biological activity of the antibody. Although the amount of antibody could also be determined using the bicinchoninic acid (BCA) method, our selective cleavage method gives more reliable results and is more powerful. Our approach provides a new direction for the component analysis of polymer nanoconjugates and nanoparticles.
doi:10.3390/ijms16048607
PMCID: PMC4425099  PMID: 25894227
polymalic acid; selective cleavage; PMLA nanoconjugate; quantitative analysis
10.  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.
doi:10.3791/50668
PMCID: PMC4118553  PMID: 24962356
Chemistry; Issue 88; Cancer treatment; personalized medicine; polymalic acid; nanodrug; biopolymer; targeting; host compatibility; biodegradability
11.  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.
doi:10.1016/j.biomaterials.2011.03.073
PMCID: PMC4110056  PMID: 21514661
12.  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.
doi:10.3791/50668
PMCID: PMC4118553  PMID: 24962356
Chemistry; Issue 88; Cancer treatment; personalized medicine; polymalic acid; nanodrug; biopolymer; targeting; host compatibility; biodegradability
13.  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.
doi:10.1016/j.jconrel.2013.06.001
PMCID: PMC3971991  PMID: 23770212
Polymalic acid; Nanobioconjugate; Nanopolymer; HER2/neu; Antibody fusion protein; IL-2; Laminin-411; Breast cancer
14.  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.
doi:10.1016/j.biomaterials.2012.08.016
PMCID: PMC3487713  PMID: 23063368
15.  Transferrin receptors and the targeted delivery of therapeutic agents against cancer 
Biochimica et biophysica acta  2011;1820(3):291-317.
Background
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.
doi:10.1016/j.bbagen.2011.07.016
PMCID: PMC3500658  PMID: 21851850
transferrin receptor; CD71; cancer; nanoparticles; immunotoxins; delivery; conjugates; gene therapy
16.  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.
doi:10.1016/j.neuroimage.2010.01.105
PMCID: PMC3524337  PMID: 20149882
Nanoplatforms; Nanotechnology; Image-guided therapy; Nanomedicine; Nanoneurosurgery; Nanostructures; Contrast agents; Nanoparticles; Nanotechnology policy; Nano-radiology; Nano-neuroscience; Nano-neurology
17.  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.
doi:10.1002/mabi.201100107
PMCID: PMC3496083  PMID: 21793213
anticancer DDS; biodegradable nanoparticles; polymalates; poly(malic acid)
18.  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.
doi:10.1016/j.ijpharm.2011.04.035
PMCID: PMC3487703  PMID: 21550387
Degradable polyesters; Poly(malic acid) derivatives; Degradable functional nanoparticles; In vitro cytotoxicity; In vitro cellular uptake
19.  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.
doi:10.1007/s10456-006-9046-9
PMCID: PMC3487708  PMID: 17109197
Tumor angiogenesis; Glioma; Laminin-8; Multiple drug targeting; Poly(malic acid)
20.  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.
doi:10.1021/bc0502457
PMCID: PMC3487710  PMID: 16536461
21.  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.
doi:10.1155/2010/825363
PMCID: PMC3459346  PMID: 23024655
22.  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.
doi:10.1371/journal.pone.0031070
PMCID: PMC3280290  PMID: 22355336
23.  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.
doi:10.3390/ijms130911681
PMCID: PMC3472769  PMID: 23109877
polymalic acid; doxorubicin; nanoconjugate; pH-controlled hydrazine linkage; brain and breast cancer
24.  Temozolomide Delivery to Tumor Cells by a Multifunctional Nano Vehicle Based on Poly(β-L-malic acid) 
Pharmaceutical research  2010;27(11):2317-2329.
Purpose
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1007/s11095-010-0091-0
PMCID: PMC2952070  PMID: 20387095
anti-TfR mAb; nanoconjugate; pH-dependent membrane disruption; polymalic acid; targeted drug delivery; temozolomide
25.  ADENOVIRUS-DRIVEN OVEREXPRESSION OF PROTEINASES IN ORGAN-CULTURED NORMAL HUMAN CORNEAS LEADS TO DIABETIC-LIKE CHANGES 
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.
doi:10.1016/j.brainresbull.2009.10.007
PMCID: PMC2815249  PMID: 19828126
diabetic cornea; organ culture; MMP-10; cathepsin F; Akt; sildenafil

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