Polymalic Acid Research Articles

Abstract 3854: Newly designed nanobioconjugate for direct targeting and systemic treatment of HER2-positive breast cancer

Abstract Objective: We have developed an efficient nanobipolymer drug delivery system Polycefin based on natural-derived biodegradable, non-toxic and non-immunogenic polymalic acid (PMLA) platform. We have shown that Polycefin was able to block brain tumor growth and angiogenesis. Our goal is to engineer the new Polycefin variant for systemic (I.V.) treatment of HER2-positive breast tumors in order to enhance the efficacy of trastuzumab/Herceptin®. Materials and Methods: For in vitro and in vivo treatment, we used (1) Herceptin, (2) Antisense oligonucleotides (AON) to HER2/neu, (3) three versions of Polycefin carrying either (3a) Herceptin alone, (3b) anti-mouse (M) and anti-human (H) transferrin receptor antibodies and AON (P-AON-TfRM+H), (3c) combination of Herceptin, anti-mouse TfR, and AON (P-AON-Herceptin-TfRM), and (4) PBS as a control. Anti-mouse TfR antibody mediated polymer transcytosis through tumor vascular endothelial cells, and Herceptin (and anti-human TfR) ensured polymer binding and internalization by tumor cells. HER2 overexpressing (BT-474, SKBR-3) and HER2 low-expressing (MDA-MB-231, MDA-MB-435) breast cancer cells were used to evaluate cell viability in vitro after treatments. Western blot analysis of cell lysates upon various treatments was used to detect HER2, phosphorylated Akt, and cleaved PARP expression. For in vivo study, BT-474 cells were inoculated S.C. into nude mice. Drugs were injected I.V. and tumor size was measured. For Polycefin distribution, drugs labeled with Alexa Fluor 680 were I.V. injected and tumors and major organs were harvested in 24 hours. Drug accumulation was analyzed by Xenogen IVIS 200 imager and subsequent confocal microscopy. Results: Polycefin variant P-AON-Herceptin-TfRM (3c) inhibited tumor cell growth in vitro significantly more than Herceptin (P<0.01 for HER2 overexpressing cells and P<0.02 for HER2 low-expressing cells). It also displayed a stronger inhibition of HER2 expression and Akt phosphorylation leading to apoptosis (cleaved PARP increase by Western blot analysis) than Herceptin alone or any other Polycefin variant (3a or 3b). In vivo imaging revealed that drug accumulation was markedly enhanced in tumors treated with P-AON-Herceptin-TfRM (3c) compared to other treatments. The same Polycefin version P-AON-Herceptin-TfRM (3c) produced the strongest reduction of tumor size compared to PBS or Herceptin (P<0.001). Summary: The leading version of nanobioconjugate with covalently attached combination of TfRM, Herceptin (anti-HER2 antibody) and anti-HER2 AON showed significant anti-tumor effect on HER2-positive breast cancer by enhancing tumor targeting, inhibition of HER2 synthesis and signaling, as well as suppression of tumor growth as compared with Herceptin treatment alone. The new version of nanoconjugate Polycefin could be promising next generation therapy for HER2-positive breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3854.

Direct tumor targeting using nanobioconjugate with a combination of monoclonal antibodies for breast cancer treatment.

Abstract Abstract #2124 Objective: Nanobioconjugate engineered based on polymalic acid (PLMA), which is derived from slime mold that is non-toxic, non-immunogenic, and biodegradable. Using combination of targeting antibodies, the nanopolymer candidates for drug delivery can efficiently reach breast cancer without affecting normal tissue in vivo. Controllable, unique drug releases tumor inhibiting agents specifically into cancer cells without affecting normal surrounding cells. Active drug blocks several cancer-specific tumor markers at the same time (Ljubimova JY, Nanomedicine 2008). We evaluated whether PMLA with covalently attached antibodies to transferrin receptor and nucleosome 2C5 reaches tumor site effectively and provides anti-tumor effect.
 Materials and Methods: Human breast cancer MDA-MB-435 and MDA-MB-231 cell lines were inoculated subcutaneously in the right thighs of nude mice. When tumors grew to 5 mm, each drug variant was injected intravenously. The nanoconjugates had different antibodies attached: non-immune IgG as a negative control (P-IgG), anti-human transferrin receptor (TfR) antibody (P-H), anti-human and anti-mouse TfR antibodies (P-H/M), and anti-tumor 2C5 antibodies (P-2C5). All drug variants were labeled with Alexa Fluor 680 tracking dye. For assessment of drug distribution at different time points and localization in various organs after intravenous injection, Xenogen IVIS 200 imager was used. The tumor and major organs were harvested after 24 hours to analyze drug distribution. Confocal microscopy of frozen sections was used for histological confirmation of drug accumulation in tissues.
 Results: Anti-mouse TfR antibody delivered nanoconjugate across the mouse endothelial system and the drug reached the tumor site specifically through anti-human TfR or anti-tumor 2C5 antibodies. The targeting efficacy of Polycefin variants was quantitatively compared against P-IgG. Weak P-IgG signal was detected in tumor probably based on non-specific enhanced permeability and retention effect. Drugs P-H and P-M/2C5 with antibodies for active targeting showed high accumulation in human tumor (p=0.01 vs. P-IgG). The best breast tumor targeting was obtained through P-H/M variant bearing a combination of anti-mouse and anti-human TfR antibodies (p=0.0001 vs. P-IgG) The drug candidate is selected to block several molecular targets specific for breast tumors treatment Her-2, EGFR or tumor endothelial marker laminin 411.
 Summary: Nanobioconjugate with a combination of anti-TfR and 2C5 antibodies showed strong tumor targeting effect in vivo. Targeting effect of antibody was more effective when the different targeting antibodies were combined than when the single antibody was used. Anti-nucleosome antibody 2C5 and TfR antibody along with polymalic acid are promising targeting tools for drug delivery. Currently, we are conducting in vivo study attaching antisense oligos/siRNA to universal nanoplatform, PLMA, to block the molecular marker such as HER-2, EGFR and laminin 411 to suppress the breast tumor growth. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2124.

Physarum polymalic acid hydrolase: Recombinant expression and enzyme activation

As a platform for syntheses of nanoconjugates in antitumor drug delivery, polymalic acid together with its tailoring specific exohydrolase is purified from plasmodium cultures of the slime mold Physarum polycephalum, a member of the phylum myxomycota. Polymalic acid hydrolase is expressed in an inactive form that functions as a molecular adapter for polymalic acid trafficking within the plasmodium and is activated only during secretion. Activation follows specific protein tyrosine phosphorylation and dissociation from plasma membranes. Purified inactive Physarum polymalic acid hydrolase, recombinantly expressed in yeast Saccharomyces, is activated on a preparative basis by the addition of plasma membrane fragments from plasmodia of P. polycephalum. Activation of polymalic acid hydrolase and inhibition of polymalic acid synthesis by protein tyrosine phosphorylation are complementary events and could indicate a joint signal response to plasma membrane damage.

Nanoconjugate based on polymalic acid for tumor targeting

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) ( 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 2.5 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.

Stage specific expression of poly(malic acid)‐affiliated genes in the life cycle of Physarum polycephalum

Polymalic acid is receiving interest as a unique biopolymer of the plasmodia of mycetozoa and recently as a biogenic matrix for the synthesis of devices for drug delivery. The acellular slime mold Physarum polycephalum is characterized by two distinctive growth phases: uninucleated amoebae and multinucleated plasmodia. In adverse conditions, plasmodia reversibly transform into spherules. Only plasmodia synthesize poly(malic acid) (PMLA) and PMLA-hydrolase (polymalatase). We have performed suppression subtractive hybridization (SSH) of cDNA from amoebae and plasmodia to identify plasmodium-specific genes involved in PMLA metabolism. We found cDNA encoding a plasmodium-specific, spherulin 3a-like polypeptide, NKA48 (spherulin 3b), but no evidence for a PMLA-synthetase encoding transcript. Inhibitory RNA (RNAi)-induced knockdown of NKA48-cDNA generated a severe reduction in the level of PMLA suggesting that spherulin 3b functioned in regulating the level of PMLA. Unexpectedly, cDNA of polymalatase was not SSH-selected, suggesting its presence also in amoebae. Quantitative PCR then revealed low levels of mRNA in amoebae, high levels in plasmodia, and also low levels in spherules, in agreement with the expression under transcriptional regulation in these cells.

Synthesis of activated poly(α,β-malic acid) using N-hydroxysuccinimide and its gelation with collagen as biomaterials

The development of synthetic biodegradable polymers, such as poly(α-hydroxy acid), is particularly important for constructing medical devices, including scaffolds and sutures, and has attracted growing interest in the biomedical field. Here, we report a synthesis method for activated polymalic acid (Su-PMA) by the reaction between synthesized α,β-PMA and N-hydroxysuccinimide (HOSu) in the presence of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC), and present an evaluation of the gelation properties of Su-PMA with collagen. The Su content of the resulting Su-PMA is higher than the feed ratio. Su content and chain length of Su-PMA influence gelation properties, as Su-PMA with low Su content formed a gel.

608 A new multifunctional drug delivery system based on polymalic acid to inhibit angiogenesis and invasion of human gliomas in vitro and in vivo

A novel bioactive lipid, cyclic phosphatidic acid (cPA), was isolated originally from myxoamoebae of a true slime mold, Physarum polycephalum, and has now been detected in a wide range of organisms from slime molds to humans. It has a cyclic phosphate at the sn-2 and sn-3 positions of the glycerol carbons, and this structure is absolutely necessary for its activities. This substance shows specific biological functions, including antimitogenic regulation of the cell cycle, regulation of actin stress fiber formation and rearrangement, inhibition of cancer cell invasion and metastasis, regulation of differentiation and viability of neuronal cells, and mobilization of intracellular calcium. Although the structure of cPA is similar to that of lysophosphatidic acid (LPA), its biological activities are apparently distinct from those of LPA. In the present review, we focus mainly on the enzymatic formation of cPA, the antimitogenic regulation of the cell cycle, the inhibition of cancer cell invasion and metastasis, and the neurotrophic effect of cPA.

Improved high temperature MSF evaporator operation for potable water production

The Royal Commission of Al Jubail and Yanbu, Kingdom of Saudi Arabia, commissioned Mitsui Engineering and Shipbuilding to design and build dual high temperature MSF evaporators to provide fresh water to the industrial city of Al Jubail. An antiscalant without polymalic acid, which is intolerant of low ferric ion concentrations, has been used successfully since startup over three years ago. Unit operating data is discussed which establishes the trouble free high temperature operation possible with antiscalant dosing. Preventive maintenance measures combined with proper system component design and choices of materials of construction have resulted in minimal corrosion and excellent performance while providing fresh water on an “as needed” basis.

Efficiency of censored reliability studies : R. J. Brooks. IEEE Trans. Reliab.R-32 (5), 504 (1983)

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.