Name: polymalic acid-based nano biopolymers for targeting of multiple tumor markers: an opportunity for personalized medicine?
Uploaded: 2014-06-13T13:00:00
Duration: 14 min 20 s
Description: Watch this Scientific Journal Video about Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine? at JoVE.com

We greatly acknowledge financial support by NIH R01 CA123495, U01 CA151815, R01 CA136841, grants from the Department of Neurosurgery at Cedars-Cedars Medical Center and Arrogene Technology Inc.

All experiments comply with official animal regulations including surgical and nonsurgical procedures performed in vivo and are in full accordance with IACUC protocols.
1. Bioproduction of Polymalic Acid
<ol>
	<li>Grow a seed culture from spherules on agar and transfer plasmodia to 100 ml culture medium using a 25 &#176;C thermo stated incubator and basal culture medium8,9.</li>
	<li>Produce an amount of gravity-packed 500 ml micro plasmodia under the exclusion of light in order to avoid sporulation.</li>
	<li>Prepare 80 g CaCO3 suspended in 8 L basal medium in 10 L bioreactor vessel. Transfer the 500 ml packed micro plasmodia into the mounted reaction vessel and allow the bioprocess for 75 hr at 25 &#176;C, 10 L/min flow of filtered air and 150 rpm stirring by a segment stirrer.</li>
	<li>Terminate when the culture broth has pH 4.8 indicating the end of production of PMLA and measure PMLA content by the hydroxamate/Fe(III) assay.</li>
	<li>Cool the broth to 17 &#176;C and allow cells to settle by gravity.</li>
	<li>Cool to 4 &#176;C and adjust to pH 7.5, use 2 M NaOH. Pump the supernatant through a column filled with 1.5 L of DEAE-cellulose in the direction bottom to top (coarse grain DEAE, equilibrated with 20 mM Tris-HCl pH 7.5 at 5 &#176;C).</li>
	<li>Wash with 3 columns of buffer containing 0.3 M NaCl after changing the direction from top to bottom and elute PMLA in the presence of 0.7 M NaCl.</li>
	<li>Adjust to 0.1 M CaCl2 and precipitate PMLA-Calcium with 80% ice cold ethanol. Fractionate over Sephadex G25 into PMLA-calcium of 80 &#8211; 300 kDa, 50 &#8211; 80 kDa, and 10 &#8211; 50 kDa, use water in the absence of buffer and salt.</li>
	<li>Pass each fraction over an Amberlite IR 120H+ column, and immediately freeze the flow-through polymalic acid in liquid nitrogen for lyophilization.</li>
	<li>Dissolve in dry acetone. After filtration, remove solvent in dry air stream, lyophilize and store at minus 20 &#176;C.</li>
</ol>
2. Synthesis of Polymalic Acid-based Nano Drug
<ol>
	<li>Activate carboxyl groups of PMLA (P) by mixing 116 mg (1 mmol malyl units) of PMLA-H in 1 ml of anhydrous acetone and 1 mmol N-hydroxysuccinimide and 1 mmol dicyclohexyl carbodimide in 2 ml dimethyl formamide (DMF). Stir at 20 &#176;C for 3 hr. Add 0.05 mmol of mPEG5000-NH2 (0.05 mol% of malyl units) in 1 ml DMF followed by 0.05 mmol triethylamine (TEA).</li>
	<li>Add drop wise dissolved in DMF 0.4 mmol leucine ethyl ester (LOEt) (40 mol% of malyl units), 0.1 mmol 2-thiol-1-ethylamine (2-MEA) (10 mol% of malyl units) and 0.5 mmol TEA, all dissolved in DMF. After each addition, allow 1 hr and check for reaction completion by negative ninhydrin response (thin layer-chromatography, TLC).</li>
	<li>Do remove leftover NHS-ester by spontaneous hydrolysis with phosphate-buffered saline (30 min, pH 6.8). Desalt over PD-10 column, obtain &#8220;preconjugate&#8221; as a white powder by lyophilization. Store dry at minus 20 &#176;C.</li>
	<li>Dissolve 30 mg antibody (mAb, IgG2a-&#954;) in 4.5 ml of 100 mM sodium phosphate, 150 mM NaCl, pH 5.5.</li>
	<li>Reduce disulfide bonds with 5 mM tris(2-carboxy ethyl)phosphine hydrochloride for 30 min at 20 &#176;C and purify over PD-10 column.</li>
	<li>Dissolve Mal-PEG3400-Mal in 2 ml of the same buffer and add the reduced mAb at final volume of 10 ml and stir overnight at 4 &#176;C. Concentrate to 2.5 ml over Vivaspin 20, exclusion 30 kD, and purify over Sephadex G75. Verify product over sec-HPLC and measure amount photometrically at 280 nm wavelength.</li>
	<li>Attach Mal-PEG-Mal-mAb to &#8220;preconjugate&#8221; thiols obtaining P/PEG5000(5%)/ LOEt(40%)/mAb(0.2%)/2-MEA(10%). Do this by mixing 5 ml of 200 nmol mAb-PEG3400-Mal to 50 mg (P/mPEG5000/LOEt/2-MEA) in the above buffer pH 5.5, adjust concentration of sulfhydryl to 2 mM and incubate at 20 &#176;C for 3 hr (yield 98%).</li>
	<li>Solubilize 3&#8217;-H2N-AON in DMF/PBS (pH 7.2) and react with N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) in (1:1 v/v) DMF/methanol (ninhydrin test) and purify AON-PDP over Sephadex-LH20 in methanol, then in water and lyophilize.</li>
	<li>Incubate 800 nmol activated AONs in the 5.5 pH buffer with the above prepared Mal-PEG3400-Mal-mAb-preconjugate overnight until the disulfide exchange reaction is terminated.</li>
	<li>Incorporate fluorescent Alexa Fluor 680-maleimide forming thioether with polymer-2-MEA-SH. Block excess sulfhydryls with 3-pyridyldithio-propionate (PDP) and purify over Sephadex G75. Store at 4 &#176;C or snap frozen at -80 &#176;C until further use.</li>
</ol>
3. Assays and Properties
<ol>
	<li>Perform tests for purity and stability in PBS and serum at 4 &#176;C and 37 &#176;C by sec-HPLC analysis. Use polystyrene sulfonate as molecular weight standards. Measure nano size distribution and zeta-potentials. Use commercial systems.</li>
	<li>Add to 320 &#956;l sample 160 &#956;l of 10% (w/v) hydroxylammonium chloride and 160 &#956;l of 10% (w/v) NaOH. After 10-15 min, mix with 160 &#956;l of 5% (w/v) Fe(III)Cl3 in 12% (v/v) HCl and read A540 of hydroxamate/Fe(III). Calculate PMLA assuming 1 mg/ml PMLA corresponds to 2.5 A540.</li>
	<li>Hydrolyze the nano drug overnight in 2 M HCl at 110 &#176;C and assay malic acid on reversed phase chromatography with reference to samples spiked with malic acid standards. Cleave the nano drug with 100 mM dithiothreitol and measure Morpholino-AON by quantitative reversed phase HPLC against Morpholino AON standards.</li>
	<li>Take nano drug without cleavage and measure antibody using a protein assay kit. Quantify mPEG by allowing its reaction with ammonium ferrothiocyanate, then extract with chloroform and read absorbance at 510 nm wavelength10.</li>
	<li>Perform ELISA with 5 &#956;l of nano drug (1-10 &#956;g mAb) per well testing antibody activity and colligation. Use 0.5 &#956;g/well transferrin receptor and HER2, respectively, as plate-coated antigens and peroxidase-coupled secondary antibodies.</li>
	<li>Calculate achieved % for AON, mPEG and mAb with regard to malic acid (100%) and compare with the % intended by design (see examples Table 1 and Table 2).</li>
</ol>
4. In vitro Testing
<ol>
	<li>Incubate the nano drug with cultured cancer cells and measure surviving cells over time by their activity to reduce yellow reagent [3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (MTS) to a blue dye and read relative absorbance with a photometer. Follow instructions by the kit supplier.</li>
	<li>Prepare cell extracts in vitro or ex vivo for Western blotting. Use ice-cold extraction buffer that contains SDS and protease inhibitor cocktail. Separate proteins by reducing SDS-polyacrylamide gel electrophoresis.</li>
	<li>Do western blotting for proteins of interest in the sample extracts. Use secondary antibodies conjugated to alkaline phosphatase.</li>
	<li>Use confocal microscopy to demonstrate cell uptake and co-localization of nano drug polymer platform, AON and endosomes using fluorescence labeling. Attach Alexa Fluor 680 to the nano conjugate platform and Lissamine to AONs as fluorescence labels. Use a microscope 5X spectral scanner and living cancer cells to visualize the distribution of the labeled molecules</li>
	<li>Stain endosome membranes with Styryl Red fluorescent dye and demonstrate endosome colocalization or release.</li>
</ol>
5. In vivo Testing
<ol>
	<li>Prepare the human HER2-positive breast cancer mouse model (nude mouse Tac:Cr:(MCr)-Foxnnm). Insert a 0.72 mg 17&#946;-estradiol releasing pellet (90-days release) subcutaneously in the neck of each mouse.</li>
	<li>Then inject 1 x 107 BT-474 tumor cells subcutaneously into the right flank and let the tumor grow.</li>
	<li>Start treatment when tumors are 120 mm3 in size by injecting into the tail vein 150 &#956;l nano conjugate containing 2.5 mg/kg AON and/or 4.5 mg/kg of each antibody. For treatment inject twice per week, totally six times.</li>
	<li>Measure tumor size with calipers twice the weak and calculate volumes using the formula (length x depth x width) x (&#960;/6). Euthanize animals 2 weeks after the final injection and after sedation with a solution of Ketamine and Dexmedetomidine followed by cervical dislocation.</li>
	<li>Isolate tumors and stain sections with H&#38;E for morphologic evaluation.</li>
	<li>For assessing drug distribution on an animal scale, use a fluorescence imaging system. Inject the Alexa Fluor 680 labeled nano drug and image at 24 and 48 hr.</li>
	<li>Euthanize the animal and detect the fluorescence in isolated and perfused tumors and organs.</li>
</ol>

<ol>
	<li>Koboldt, D. C., Fulton, R. S., McLellan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+portraits+of+human+breast+tumors.+Cancer+Genome+Atlas+Network.">Comprehensive molecular portraits of human breast tumors. Cancer Genome Atlas Network.</a> Nature. 490, 61-70 (2012).</li><li>Kwong, L. N., Costello, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogenic+NRAS+signaling+differentially+regulates+survival+and+proliferation+in+melanoma.">Oncogenic NRAS signaling differentially regulates survival and proliferation in melanoma.</a> Nat. Med. 18, 1503-1510 (2012).</li><li>Gerlinger, M., Rowan, A. J., Gerlinger, M., Rowan, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumor+heterogeneity+and+branched+evolution+revealed+by+multiregion+sequencing.">Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.</a> N. Engl. J. Med. 366, 883-892 (2012).</li><li>Yap, T. A., Workman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+the+cancer+genome:+strategies+for+the+discovery+and+clinical+development+of+targeted+molecular+therapies.">Exploiting the cancer genome: strategies for the discovery and clinical development of targeted molecular therapies.</a> Annu. Rev. Pharmacol. Toxicol. 52, 549-573 (2012).</li><li>Chatterjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Biomarkers:+Knowing+the+Present+and+Predicting+the+Future.">Cancer Biomarkers: Knowing the Present and Predicting the Future.</a> Future Oncol. 1, 37-50 (2005).</li><li>Suh, K. S., Sarojini, S., Youssif, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Banking,+Bioinformatics,+and+Electronic+Medical+Records:+The+Front-End+Requirements+for+Personalized+Medicine.">Tissue Banking, Bioinformatics, and Electronic Medical Records: The Front-End Requirements for Personalized Medicine.</a> J. Oncology. , (2013).</li><li>Ljubimova, J. Y., Holler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatible+nanopolymers:+the+next+generation+of+breast+cancer+treatment.">Biocompatible nanopolymers: the next generation of breast cancer treatment.</a> Future Medicine, Nanomedicine. 7, 1-4 (2012).</li><li>Lee, B. -. S., Vert, M., Holler, E., Steinb&#252;chel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Water-soluble aiphatic polyesters: poly(malic acid)s. Biopolymers 3a. , 75-103 (2002).</li><li>Lee, B. -. S., Fujita, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycefin,+a+new+prototype+of+multifunctional+nanoconjugate+based+on+poly(beta-L-malic+acid)+for+drug+delivery.">Polycefin, a new prototype of multifunctional nanoconjugate based on poly(beta-L-malic acid) for drug delivery.</a> Bioconjugate Chem. 17, 317-326 (2006).</li><li>Nag, A., Mitra, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+colorimetric+assay+for+estimation+of+polyethylene+glycol+and+polyethylene+glycolated+protein+using+ammonium+ferrothiocyanate.">A colorimetric assay for estimation of polyethylene glycol and polyethylene glycolated protein using ammonium ferrothiocyanate.</a> Analytical Biochem. 237, 224-231 (1996).</li><li>Ding, H., Inoue, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+brain+tumor+growth+by+intravenous+poly(beta-L-malic)+acid+nanobioconjugate+with+pH-dependent+drug+release.">Inhibition of brain tumor growth by intravenous poly(beta-L-malic) acid nanobioconjugate with pH-dependent drug release.</a> Proc. Natl. Acad. Sci. USA. 107, 18143-18148 (2010).</li><li>Inoue, S., Ding, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanobioconjugate+inhibition+of+HER2/neu+signaling+and+synthesis+provides+efficient+mouse+breast+cancer+treatment.">Nanobioconjugate inhibition of HER2/neu signaling and synthesis provides efficient mouse breast cancer treatment.</a> Cancer Research. 71, 1454-1464 (2011).</li><li>Maeda, H., Sawa, T., Konno, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+tumor-targeted+delivery+of+macromolecular+drugs,+including+the+EPR+effect+in+solid+tumor+and+clinical+overview+of+the+prototype+polymeric+drug+SMANS.">Mechanism of tumor-targeted delivery of macromolecular drugs, including the EPR effect in solid tumor and clinical overview of the prototype polymeric drug SMANS.</a> J. Control. Release. 74, 47-61 (2001).</li><li>Ljubimova, J. Y., Fujita, M., Ljubimov, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(malic+acid)+nanoconjugates+containing+various+antibodies+and+oligonucleotides+for+multitargeting+drug+delivery.">Poly(malic acid) nanoconjugates containing various antibodies and oligonucleotides for multitargeting drug delivery.</a> Nanomed. 3, 247-265 (2008).</li><li>Inoue, S., Patil, R., Portilla-Arias, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+nanobioconjugate+inhibiting+EGFR+expression+in+triple+negative+breast+cancer.">Novel nanobioconjugate inhibiting EGFR expression in triple negative breast cancer.</a> PLoS One. 7, 1-9 (2012).</li><li>Patil, R., Portilla-Arias, J., Ding, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Delivery+of+Doxorubicin+via+pH-Controlled+Hydrazone+Linkage+Using+Multifunctional+Nano+Vehicle+Based+on+Poly(&#946;-L-malic+Acid).">Cellular Delivery of Doxorubicin via pH-Controlled Hydrazone Linkage Using Multifunctional Nano Vehicle Based on Poly(&#946;-L-malic Acid).</a> Int. J. Mol. Sci. 13, 11681-11693 (2012).</li><li>Patil, R., Portilla-Arias, J., Ding, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temozolomide+delivery+to+tumor+cells+by+a+multifunctional+nano+vehicle+based+on+poly(&#946;-L-malic+acid).">Temozolomide delivery to tumor cells by a multifunctional nano vehicle based on poly(&#946;-L-malic acid).</a> Pharm. Res. 27, 2317-2329 (2010).</li><li>Ljubimova, J. Y., Portilla-Arias, J., Patil, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+and+efficacy+evaluation+of+multiple+targeted+polymalic+acid+conjugates+for+triple-negative+breast+cancer+treatment.">Toxicity and efficacy evaluation of multiple targeted polymalic acid conjugates for triple-negative breast cancer treatment.</a> J. Drug Target. 21, 956-967 (2013).</li></ol>

The authors Julia Y. Ljubimova, Keith L. Black, and Eggehard Holler are shareholders of Arrogene Technology Inc.

The experimental route for the preparation of nano drugs from a biodegradable natural polymer is presented that can be used in the synthesis of personalized medicine. The description starts with the controlled production and purification of polymalic acid, which is a versatile platform for nano drug synthesis. Using reproducible techniques, the polymer is obtained with high molecular weight and in extreme purity suitable for pharmaceutical syntheses. The synthesis is described for a nano drug that is shown to efficiently treat HER2-positive breast cancer. The description can be translated into syntheses of most other nano drugs for the treatment of cancer. Targeting involves antibodies such as Herceptin that bind a tumor specific antigen such as HER2 protein or any other tumor marker that is efficiently internalized. The nano drug delivers a selection of antisense oligonucleotides and chemotherapeutics that will efficiently inhibit growth of the cancer under treatment. In the example, Herceptin binding to HER2 and specific antisense oligonucleotide annealing with HER2-coding mRNA resulted in sustained blocking of HER2-signaling and severe reduction of HER2-positive breast cancer. Based on the underlying principle of tumor targeting and inhibition of gene expression by antisense oligonucleotides we have to-date synthesized several other nano drugs and successfully inhibited preclinical human glioblastoma and triple-negative breast cancer11,14-18.
The synthetic work starts with the preparation of highly purified nano drug platform, which is polymalic acid from the culture supernatant of Physarum polycephalum (a species of the &#8220;slime mold&#8221; family). The preparation emphasizes high molecular weights of the polymer, in principle allowing attachment of numerous antibodies, peptides, oligonucleotides and other molecules functioning in active drug delivery and tumor growth inhibition. Following the controlled culturing and purification, reproducible quality of polymer in predictable yields has been produced. The polymer is stored under convenient conditions for any time.
The synthesis of PMLA nano conjugates beginning with the chemical activation of the polymer-pendant carboxylates is carried out in a few synthetic steps. In between steps, synthesis can be optionally placed on hold allowing preparation of arbitrary amounts of intermediates and thus can be used in up scaling. The progress of synthesis is followed by TLC and sec-HPLC, and both the composition and activity of the nano drug is controlled by group-specific quantitative chemical assays, ELISA, and a variety of physical measurements. Our experience is that these syntheses have been progressed smoothly and reproducibly with excellent yields and purity. By choosing the specificity of antibody and antisense oligonucleotides any variant of nano drug is favorably synthesized as needed in personalized medicine.
The modalities of the successful treatment of human HER2-positive breast cancer are valid representatives for preparation of mouse cancer models, application of nano drugs, imaging, and analysis of tumor growth. The results of in vitro viability tests are useful in selecting the cell line and the leading drug to be used in the animal experiment. In vivo Xenogen imaging validates that the nano drug is indeed delivered into the tumor. Results of western blotting reveals whether the level of certain proteins responded in the predicted fashion during cancer treatment. Measurement of tumor size informs about the success of the treatment, i.e. about inhibition, recession or regression. The described example reflects our experience with other polymalic acid-based nano drugs indicating a high degree of predictability, reproducibility and absence of notable toxicity. Recent results on toxicity and efficacy of multiple targeted polymalic acid conjugates for triple-negative breast cancer treatment are in strong support of this notion18. A key feature is that our nano drug is able to penetrate bio-barriers: the endothelial barrier by extravasation into the tumor interstitial, the tumor cell membrane by endosome uptake, and endosome membrane disruption by action of the leucine ethylester or trileucine groups. Extravasation and endosome uptake are reliably accomplished by the specific antibodies attached to the nano drug. Anti-mouse transferrin receptor antibody mediates efficient influx into the tumor by transcytosis, probably because the receptor is overexpressed on most tumor vasculature. A different antibody attached on the same nano conjugate molecule functions in specifically directing the nano drug into the recipient tumor cell. The presence of both antibodies, the one for transcytosis and the other one for endosome uptake, is essential for optimal functioning. Quantitative analysis of malic acid and antibody is highly recommended in order to control optimal composition of the nano drug. Finally it should be noted that the all-in-one covalent nano drug delivers drug in chemically attached form en route through the host vasculature into the recipient tumor cell. Chemical attachment renders most drugs inactive (prodrugs) until they are reconstituted as free drugs by cleavage from the nano conjugate platform at the targeted site. This is important because the reactivation modality provides a high degree of safety during delivery and a minimal chance to evoke harmful side effects. Versatility, efficacy and safety are indispensable attributes of good personalized medicine.

In the post-genomic era when genomes of cancer have been unraveled (National Center for Biotechnology and The Cancer Genome Atlas), future treatment of cancer will account for genetic diversity of tumors often within the same tumor1-4. Bioinformatics and fast, not expensive DNA sequencing allows acquisition of malignant genes/mutations on a personal level2,4,5. Once the genes have been identified, patients will be treated with personalized medicine to modify or silence their expression of malignant genes6. The need to target cancer cells and deliver drugs into these cells calls for polyfunctional delivery systems. Obviously, nano drugs can meet this requirement7.
In a surging wave of discoveries nanoparticles have proven suitable to bring payloads of chemotherapeutic drugs, proteins and/or genetically active material to cancer cells. However, adverse effects remain to be addressed. One of them related to the absence of biodegradability may cause deposition of material in healthy tissue and organs with the likelihood to provoke diseases. To minimize deposition, we introduced non-toxic and non-immunogenic polymalic acid, which is of microbial origin and biodegradable to H2O and CO28. We use the polymer to synthesize an all-in-one covalent type of nano drug. It contains chemically attached chemotherapeutics such as Temozolomide, Doxorubicin, or antisense oligonucleotides and functional groups serving extravasation, tissue targeting, endosomolytic delivery. The drugs are intrinsically cleaved from the nano platform when they arrived in the targeted tumor cell, thereby regenerating their full pharmaceutical activity.
We describe the method of microbial production of the polymer nano platform, its purification, and the chemical synthesis of a nano drug that contains Trastuzumab (Herceptin) for cancer targeting and an antisense oligonucleotide for the inhibition of HER2 overproduction. In applying the nano drug to xenogeneic human Her2-positive breast cancer on nude mice, we demonstrate high efficacy of the cancer treatment. The principles of tumor targeting and gene silencing introduced here for polymalic acid nano drugs can be applied in the treatment of other cases of cancer.

<table border="0" cellpadding="0" cellspacing="0" width="935">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Name of Reagent/Material</td>
			<td style="width:263px;">Company</td>
			<td style="width:187px;">Catalog Number</td>
			<td style="width:79px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercapto-1-ethylamine (Cysteamine (2MEA)</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">30078-25G</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8217;,6-Diamidino-2-phenylindole (DAPI)</td>
			<td colspan="2">Vector Laboratories, Burlingame, CA</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">90-Day release 17&#946;-estradiol pellet&#160;</td>
			<td>Innovative Research of America</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 680 C2 maleimide</td>
			<td style="width:263px;">Invitrogen</td>
			<td style="width:187px;">A20344</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amberlite IR 120H</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">6428</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Antifoam Y-30 Emulsion</td>
			<td style="width:263px;">Sigam-Aldrich</td>
			<td style="width:187px;">A5758</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Anti-laminin-411 chain mAbs</td>
			<td style="width:263px;">Santa Cruz</td>
			<td style="width:187px;">SC-59980</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Anti-pAkt mAb</td>
			<td style="width:263px;">Cell Signalling</td>
			<td style="width:187px;">9271S</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Anti-PARB mAb</td>
			<td style="width:263px;">BD Biosciences</td>
			<td style="width:187px;">556494</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-von Willebrand Factor antibody&#160;</td>
			<td>Abcam</td>
			<td style="width:187px;">AB6994</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Bacto Yeast Extract</td>
			<td style="width:263px;">Bacto, Dickinson/Sparks, MD</td>
			<td style="width:187px;">212720</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beta-actin</td>
			<td style="width:263px;">Cell Signalling</td>
			<td style="width:187px;">3700</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BT-474&#160;</td>
			<td style="width:263px;">ATCC</td>
			<td style="width:187px;">HTB-20</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Calcium Carbonate</td>
			<td style="width:263px;">Alfa Aesar</td>
			<td style="width:187px;">36337</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Proliferation Assay kit</td>
			<td>Promega, Madison, WI, USA</td>
			<td>PR-G3580</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centriplus 100&#160;</td>
			<td>Millipore</td>
			<td style="width:187px;">4414</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dicyclohexylcarbodimide</td>
			<td style="width:263px;">Fluka</td>
			<td style="width:187px;">36650</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">D5796</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eosin</td>
			<td style="width:263px;">Cardinal Health</td>
			<td style="width:187px;">S7439-4</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Galardin (MMP-inhibitors)&#160;</td>
			<td style="width:263px;">Santa Cruz</td>
			<td style="width:187px;">SC-994</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GAPDH</td>
			<td style="width:263px;">Cell Signalling&#160;</td>
			<td style="width:187px;">2118</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematoxilin</td>
			<td style="width:263px;">Cardinal Health</td>
			<td style="width:187px;">S7439-3</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Hemin from porcine</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">51280</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Herceptin</td>
			<td style="width:263px;">Genentech</td>
			<td style="width:187px;">15534</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Herceptin (Western blotting)</td>
			<td style="width:263px;">Cell Signalling</td>
			<td style="width:187px;">2165S</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">IgG2a-kappa murine malignoma</td>
			<td style="width:263px;">Sigma</td>
			<td style="width:187px;">M77695X5m</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immun-Star AP Substrate Pack</td>
			<td style="width:263px;">Biorad</td>
			<td style="width:187px;">170-5012</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immun-StarTM AP Substrate Pack&#160;</td>
			<td>Biorad</td>
			<td>170-5012</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">LAL Reagent water&#160;</td>
			<td style="width:263px;">Lonza, MD</td>
			<td style="width:187px;">W50-1000</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin-411 mAbs</td>
			<td style="width:263px;">Abcam</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leucine ethyl ester (LOEt)</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">61850-10G-F</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Limulus Amebocyte Lysate (LAL) PYROGENT-5000 tests</td>
			<td style="width:263px;">Cambrex BioScience</td>
			<td style="width:187px;">N384</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Lissamin-Morpholino AON</td>
			<td style="width:263px;">Gene Tools</td>
			<td style="width:187px;">Custom made</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-malic acid&#160;</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">M6413</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Malate dehydrogenase&#160;</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">M2634</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mal-PEG-Mal</td>
			<td style="width:263px;">Laysan</td>
			<td style="width:187px;">mal-PEG-mal-3400</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel</td>
			<td>BD Biosciences</td>
			<td style="width:187px;">354248</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Milk, nonfat powdered&#160;</td>
			<td style="width:263px;">Proteomics</td>
			<td>M203-10G</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Morpholino oligonucleotides&#160;</td>
			<td style="width:263px;">GeneTools</td>
			<td style="width:187px;">custom made</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">mPEG5000</td>
			<td style="width:263px;">Lysan</td>
			<td style="width:187px;">mPEG-NH2-5000</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-hydroxysuccinimde (NHS)&#160;</td>
			<td style="width:263px;">ACROS Organics</td>
			<td style="width:187px;">15727100</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Ninhydrin</td>
			<td style="width:263px;">Merck</td>
			<td style="width:187px;">1.06762.0100</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose Membrane Filter Paper Sandwich</td>
			<td>Invitrogen</td>
			<td>LC2001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose Membrane Filter Paper Sandwich, 0.45 &#181;m&#160;</td>
			<td style="width:263px;">Invitrogen</td>
			<td style="width:187px;">LC2001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Novex &#174; Tris-Glycine Transfer Buffer (25X)&#160;</td>
			<td>Invitrogen</td>
			<td>LC3675</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Nude Mice [Tac:Cr:(MCr)-Foxnnm]&#160;</td>
			<td>Taconic</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">PBS pH 7.2 10x</td>
			<td style="width:263px;">Gibco</td>
			<td style="width:187px;">10010-49</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">PBS pH 7.2 1x</td>
			<td style="width:263px;">Gibco</td>
			<td style="width:187px;">70013</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PD-10 desalting columns</td>
			<td style="width:263px;">GE Healthcare</td>
			<td style="width:187px;">17-0851</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Physarum polycephalum M3CVII</td>
			<td>ATCC</td>
			<td>204388</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce BCA protein assay&#160;</td>
			<td style="width:263px;">Thermo Scientific</td>
			<td style="width:187px;">23225</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease inhibitor cocktail</td>
			<td style="width:263px;">Roche</td>
			<td style="width:187px;">1.18362E+11</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein Detector ELISA Kit</td>
			<td style="width:263px;">KPL</td>
			<td style="width:187px;">54-62-18</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:263px;"></td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex G-25 superfine</td>
			<td style="width:263px;">GE Healthcare</td>
			<td style="width:187px;">17-0031</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex G-75&#160;</td>
			<td style="width:263px;">GE Healthcare</td>
			<td style="width:187px;">17-0050</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex-LH20&#160;</td>
			<td style="width:263px;">GE Healthcare</td>
			<td style="width:187px;">17-0090</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SKBR-3&#160;</td>
			<td style="width:263px;">ATCC</td>
			<td style="width:187px;">HTB-30</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPDP</td>
			<td style="width:263px;">Proteochem&#160;</td>
			<td style="width:187px;">C1116</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streamline-DEAE</td>
			<td style="width:263px;">GE Healthcare</td>
			<td style="width:187px;">17-0994</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Styryl Red FM 1-43&#160;</td>
			<td>Life Technologies&#160;</td>
			<td style="width:187px;">T-3163</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:263px;"></td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TBS 10x</td>
			<td>Bio-Rad</td>
			<td>170-6435</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TCEP</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">C4706-2G</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TLC, silica coated aluminia sheets</td>
			<td style="width:263px;">Merck, Darmstadt, DE</td>
			<td style="width:187px;">60F254</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trileucine (LLL)</td>
			<td style="width:263px;">Bachem</td>
			<td style="width:187px;">H-3915</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-114&#160;</td>
			<td style="width:263px;">Sigma-Aldrich</td>
			<td style="width:187px;">X114</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween&#174;20&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:263px;"></td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vivaspin 20&#160;</td>
			<td style="width:263px;">VWR</td>
			<td style="width:187px;">14005-302</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:407px;">DAPI</td>
			<td style="width:263px;">Vector Laboratories, Burlingame, CA</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dexmedetomidine</td>
			<td style="width:263px;">Pfizer</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Atipamezole</td>
			<td style="width:263px;">Pfizer</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carprofen</td>
			<td style="width:263px;">Pfizer</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine</td>
			<td style="width:263px;">Foster and Smith, Wisconsin</td>
			<td style="width:187px;"></td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

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.

Inhibition of human HER2-positive breast cancer by silencing HER2 receptor expression and blocking HER2-signaling12
Strategy
Among different forms of human breast cancer, HER2-positive tumors have the worst clinical outcome. We present the successful treatment of human HER2-overexpressing breast cancer in the nude mouse model. The strategy involves the nano drug active in both the immediate silencing of the HER2 signaling pathway employing Herceptin and HER2-specific AON for blocking the HER2-mRNA dependent synthesis of the receptor (Figure 1). The lead nano conjugate, which contains Herceptin and anti-HER2 AON is shown in Figure 3 together with two controls which are devoid of either Herceptin or AON. The lead compound contained anti-MsTfRmAb to achieve active extravasation by transcytosis through binding to endothelial TfR of the tumor vessels. Much less uptake into the tumor was noted in the absence of the antibody and was attributed to tumor dependent EPR effect13. Fluorescent versions of the nano conjugates containing Alexa Fluor 680 were synthesized for imaging studies.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/50668/50668fig1highres.jpg" width="500" /> 
Figure 1. Mechanism of AON delivery into HER2-positive breast cancer cells and tumor growth inhibition. Upper left corner: Nano conjugate adapted from Figure 3. Lower left corner: Mouse tumor vessels expressing TfR on the surface. The nano drug binds to the receptor and enters the tumor by transcytosis. In addition, some degree of access to the tumor is possible through the disordered endothelial layers that participate in tumor dependent enhanced uptake and retention (EPR)13. Next, the nanodrug binds to HER2 expressed on the human cancer cells and internalizes into early endosomes. Binding also blocks HER2 signaling pathway. After maturation of endosomes and their acidification, the endosome escape mechanism of the nano drug is activated. Nanodrug entering the cytoplasm is released from the nano platform by reductive cleavage of the disulfide spacer and binds to HER2-mRNA blocking HER2-synthesis. Blocking of the synthesis extends blocking of HER2-signaling and induces tumor growth inhibition.
Microbial production of the polymalic acid platform
The nano conjugate platform, polymalic acid (PMLA), was produced by cultured microplasmodia of Physarum polycephalum and purified from the broth as described in the Protocol and depicted in Figure 2. Production and purification were smooth and reproducible. It was important to control time, pH and temperature to avoid spontaneous cleavage of the polymer. Careful washing and elution of the DEAE-column is recommended in order to remove DNA, carbohydrates, toxins and colored material. Extreme purity was obtained after dissolving in anhydrous acetone and removing insoluble material. Total amount of produced PMLA was 5 &#177; 1 g per campaign. Amounts of 1.5 &#177; 0.5 g highly purified PMPLA of Mw 80,000-100,000 were reproducibly achieved when using the 10 L bioreactor. Sec-HPLC elution profiles were symmetrical. Dynamic light scattering analysis according to number distribution indicated a single peak corresponding to a hydrodynamic diameter of 8 &#177; 1 nm and a polydispersity index of PDI = 0.10 &#177; 0.02, calculated by the instrument software for assumed spherical particles. Zeta-potential was -22 &#177; 2 mV (pH 7.5).
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/50668/50668fig2highres.jpg" width="500" /> 
Figure 2. Production and purification of polymalic acid from cultured micro plasmodia of Physarum polycephalum (adapted from Lee et al.9). Polymalic acid (PMLA) is produced from plasmodia of Physarum polycephalum in a bioreactor and collected from the culture supernatant by binding on anion exchanger (DEAE). The eluent is ethanol-precipitated as calcium salt. Solutions of the calcium salt are Mw-fractionated over Sephadex-G25. PMLA containing fractions are converted from the Ca-salt into the acid over Amberlite IR-120H+. The free PMLA is finally lyophilized to yield dry white powder.
Chemical synthesis of the lead nano conjugate 
P/mPEG5000(5%)/LOEt(40%)/AON(2.5%)/Herceptin(0.2%)/anti-MsTfRmAb(0.2%)
The schematic structures of the lead nano drug and of two precursor nano conjugates are shown in Figure 3. The lead contains all constituents, while the precursors were designed to lack either AONHER2 or the antibody Herceptin. Besides AON and mAbs, the compounds contained polyethylene glycol, mPEG5000, to minimize binding to plasma proteins, clearance through the reticulo-endothelial system (RES), and degradation by enzymatic cleavage. The antisense sequence 5&#8217;-CAT-GGT-GCT-CAC-TGC-GGC-TCC-GGC-3&#8217; was complementary to Her2 mRNA and has been carefully tested in vitro on several HER2-expressing cell lines to obtain high specificity towards blocking HER2 synthesis and not showing off-target effects.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/50668/50668fig3highres.jpg" width="500" /> 
Figure 3. Nano conjugates to treat human HER2-positive breast cancer. Leading nano drug (top structure) contains two drugs (Herceptin, AONHER2). Versions 2 and 3 contain only one of the drugs. Uptake of 1 and 2 into human HER2-overexpressing tumor cells is managed by the binding of Herceptin. Nano conjugate 3, which does not contain Herceptin, received anti-HuTfRmAb for endosome uptake by the human cells. The conjugation with Alexa Fluor 680 was optional for imaging purposes. The red bar represents the nano conjugate platform PMLA/LOEt(40%). % indicates the fraction of malic acid residues consumed in binding of the indicated ligand (total amount of malic acid residues in the nanodrug = 100%).
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/50668/50668fig4highres.jpg" width="500" /> 
Figure 4. Chemical synthesis of nano conjugates P/LOEt/AONHER2/anti-MsTfR/Herceptin. From top to bottom: Synthesis of PMLA-N-hydroxy succinimidyl ester (PMLA-NHS) of pendant carboxylates (chemical activation). Replacement of NHS by amide formation with 2-mercapto-1-aminoethan (2-MEA), methyl-PEG5000-amine (mPEG5000-NH2), trileucine (LLL) or leucine ethylester (LOEt). This &#8220;preconjugate&#8221; attaches mAb-Mal-PEG-Mal by thioether formation and AON by formation of cleavable disulfide bonds. Leftover 2-MEA is capped forming amidoethyl-dithio-propionic acid. Only attachment of a single mAb is shown. Multiple attachments are managed by using mixtures of mAbs. Percentage % denotes fractions of carboxylates bound to various ligands (100% for free PMLA).
The nano conjugates were synthesized as outlined in Figure 4. The calculated molecular weight of the lead conjugate was 719,000. The overall yield of the synthesis was 45 &#177; 5% with regard to the malic acid content. On average, of the 862 malyl units (=100%) of the 100 kDa PMLA platform, 40% carried LOEt, 5% mPEG, 2% AON 2%, 0.2% Herceptin and 0.2% anti-MsTfR mAb. The amount for each antibody corresponded to an average of 1.7 molecules per molecule of PMLA platform. The % designed and the % verified by group analysis were the same within &#177;5%. An example is the case given in Table 1 for the calculation of the experimental content of malic acid, AON, mAb and mPEG5000 and in Table 2 shows the comparison with the content by design. The high purity according to the criteria under section 3 was achieved on the basis of high reaction yields and efficient separation by size exclusion (e.g. free mAb and mAb-nanoconjugate are separated by 1 min on sec-HPLC), and selective solubility in solvents. The activity of monoclonal antibodies was shown to be retained throughout nano drug synthesis, and it was confirmed that the two kinds of antibodies were assembled on the same polymer platform. The result of atypical ELISA experiment is shown in Figure 5. In general, the assays for quantitative group analysis for malic acid, AON, protein, PEG and for ELISA were robust, yielding reproducible results when conducted by different personnel and instrumentation not only in the case of the reported synthesis, but also when used for analysis of other synthesized nanoconjugates.
<img alt="Table 1" fo:content-width="6in" src="/files/ftp_upload/50668/50668table1.jpg" width="600" /> 
Table 1. Calculation of nanodrug composition with reference to malic acid content.
<img alt="Table 2" fo:content-width="6in" src="/files/ftp_upload/50668/50668table2.jpg" width="600" /> 
Table 2. Comparison of experimental nano drug composition with designed composition.
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/50668/50668fig5highres.jpg" width="500" /> 
Figure 5. ELISA for measurement of antibody affinity after chemical synthesis, and for demonstration of multiple binding of different antibodies. The nano drug contains antibody mAb(A) and antibody mAb(B) against antigens A and B, respectively. ELISA plates are coated with antigen(A). Free mAb(A), free mAb(B), and nano drug are applied to ELISA plates. After washing, the antibodies are tested with secondary peroxidase coupled antibodies specific for either mAb(A) or mAb(B), while only mAb(A) is bound to antigen(A). It is seen that free and nano drug conjugated with mAb(A), and indirectly conjugated mAb(B) of the same nano drug molecule, but not free mAb(B), are retained on the plate. The concentration dependence shows comparable binding affinities for free and conjugated mAb(A), indicating that the chemical conjugation did not affect antibody binding activity. Moreover, the co-ligation of mAb(B) with mAb(A) on the same physical entity (nano platform) results in antibody mAb(B) detection. The experimental results shown here refer to anti-human TfRmAb (A) and anti-mouse TfRmAb (B). Similar results have been shown for other antibody couples tested, such as anti-MsTfR mAb and anti-HER2 mAb (Herceptin).
The physicochemical investigation indicated hydrodynamic diameter 22.1 &#177; 2.3 nm for P/mPEG(5%)/LOEt(40%)/AON(2%)/Herceptin(0.2%)/anti-MsTfRmAb(0.2%) (lead, the two-drug version), 20.1 &#177; 2.4 nm for P/mPEG(5%)/LOEt(40%)/AON(2%)/anti-MsTfRmAb(0.2%)/anti-HuTfRmAb (0.2%) (the AON drug version), and 15.1 &#177; 1.2 nm for P/mPEG(5%)/LOEt(40%)/Herceptin(0.2%) (the Herceptin drug version). Zeta-potentials at pH 7.5 were in this order -5.2 &#177; 0.4 mV, -5.7 &#177; 0.6 mV, and -4.1 &#177; 0.4 mV. It should be mentioned that the measured hydrodynamic diameter of nano conjugates did not follow additivity of the diameters measured for free components.
Delivery of nano drug through the tumor cell membrane and release into the cytoplasm after 0 hr and 3hr
The delivery of nano drug through the cell membrane by endosome uptake is shown in Figure 6 after 0 hr and 3 hr. Fluorescence labels are attached to the nano drug platform (Alexa Fluor 680, green) and to AON (Lissamine, red). Their superposition is shown in panels D&#38;L and together with the fluorescence of labeled endosomes (blue) in panels G&#38;O. Pearson&#8217;s correlation coefficients (R(r) were calculated from the images regarding co-localization of platform/endosomes, AON/endosomes, platform/AON for 0 hr and 3 hr. The 3 hr image indicated release from endosomes into the cytoplasm and dissociation of AON from the nano drug by glutathione-dependent disulfide cleavage in the cytoplasm.
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/50668/50668fig6highres.jpg" width="500" /> 
Figure 6. Confocal microscopy for nano conjugate uptake by target cells (adapted from Ding et al.11). Cells were incubated for 30 min at 37 &#176;C with nano drug which had been double labeled with Alexa Fluor 680 (green) at the polymer platform and with Lissamine (red) attached to AON. In addition, endosomes were stained with FM1- 43 (blue). Localization is shown after 0 hr (A) and 3 hr (B) incubation. Panels A,a-b, &#38; B,i-j show staining by the nano conjugate fluorophores alone, and their co-localization in A,c-d &#38; B,k-l. Additionally, staining of endosomes is shown in panels A,e &#38; B,m and the colocalization of nano conjugate and endosomesin panels A,f-g &#38; B,n-o. Panels A,h &#38; B,p show phase contrast for respective panels. (C) Pearson&#8217;s correlation coefficients R(r) for the colocalization of platform-endosomes, AON-endosomes, platform-AON calculated for 0 hr and 3 hr. <a href="https://www.jove.com/files/ftp_upload/50668/50668fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Inhibition of human breast cancer in vitro and in vivo 
In vitro growth inhibition of HER2 overexpressing cell line BT-474 in comparison with low expressing cell line MDA-MB-231 is shown in Figure 7. Degree of inhibition is 50% and 30%, respectively, by the lead nano conjugate P/mPEG/LOEt/AONHER2/Herceptin/anti-MsTfRmAb. Inhibition by the other compounds is less but higher for BT-474 than for MDA-MB-231 cells. BT-474 cell line was chosen for the treatment of xenogeneic breast cancer mouse.
<img alt="Figure 7" fo:content-width="5in" src="/files/ftp_upload/50668/50668fig7highres.jpg" width="500" /> 
Figure 7. In vitro growth inhibition of HER2 overexpressing BT-474 breast cancer cells and low expressing MDA-MB-231 cells (adapted from Inoue et al.12). Comparison of growth inhibition for HER2 overexpressing BT-474 breast cancer cell line (left) and of HER2 low expressing breast cancer cell line MDA-MB-231 (right). TfR(s) denotes anti-MsTfRmAb and TfR(Hu/Ms) denotes anti-HuTfRmAb &#38; anti-MsTfRmAb. The lead nano drug, P/mPEG/LOEt/AONHER2/Herceptin/TfR(Ms), and nano drugs devoid of either Herceptin or AONHER2 are compared with PBS, Herceptin and AONHER2. In the absence of Herceptin, anti-MsTfRmAb was conjugated to provide endosome uptake by the tumor cells. Concentrations were 40 &#956;g/ml with regard to antibodies, 4 &#956;M with regard to AONHER2, 4 &#956;M endoporter (application of AON). Significance denoted by *, P&#60;0.05: **, P&#60;0.02: ***, P&#60; 0.003 compared with PBS.
The results of in vivo treatment in Figure 8 are presented for mice bearing BT-474 Her2 overexpressing human breast cancer. Growth was inhibited &#62;95% by the lead nanodrug containing Herceptin and HER2-specific AON in comparison to controls treated only with PBS (Figure 8). Inhibition was 60% or less with Herceptin alone, P/mPEG/LOEt/Herceptin or P/mPEG/LOEt/AON/anti-MsTfRmAb/anti-HuTfRmAb. Western blot analysis of tumor extracts showed inhibition of both HER2 synthesis and Akt phosphorylation, but no change of total Akt and housekeeping enzyme GAPDH. PARP was cleaved in correspondence with an increased level of apoptosis. Pictures of tumor after treatment, and tissue sections stained with H&#38;E illustrating tumor regression are shown in Figure 9.
<img alt="Figure 8" fo:content-width="5in" src="/files/ftp_upload/50668/50668fig8highres.jpg" width="500" /> 
Figure 8. Tumor size and proteins during treatment with lead and precursor nano drugs (adapted from Inoue et al.12). A. Tumor size for the various treatments (day 21 to day 42, totally 8 injections). Bars indicate standard deviations from the mean. The lead conjugated with anti-Her2 AON and Herceptin was superior to either Herceptin alone or single drug containing nano conjugates. B. Effect of the various treatments on the expression of HER2, phosphorylated Akt, total Akt, PARB, cleaved PARB and GAPDH are shown by western blotting. <a href="https://www.jove.com/files/ftp_upload/50668/50668fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" fo:content-width="5in" src="/files/ftp_upload/50668/50668fig9highres.jpg" width="500" /> 
Figure 9. Treatment of HER2-positive BT-474 human breast cancer on nude mice. Tumor regression and necrosis/apoptosis in tissue sections (adapted from Ionue et al.12). Upper: Shrinkage of the tumor (red arrows). Indication of the estrogen pellet to support tumor growth (blue arrow). Lower: Hematoxilin &#38; Eosine (H&#38;E) staining of tumor sections. Proliferating tumor is shown by the dense packing of tumor cells. Necrotic tissue is seen where tumor cells have been eliminated. <a href="https://www.jove.com/files/ftp_upload/50668/50668fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine? at JoVE.com

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

An example of a nano drug based on polymalic acid is presented towards the rational design of personalized medicine that is applicable to cancer. It describes the synthesis of a nano drug to treat Her2-positive human breast cancer in a nude mouse.

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

polymalic-acid-based-nano-biopolymers-for-targeting-multiple-tumor

Research

JoVE Journal

Chemistry

Glycan Node Analysis: A Bottom-up Approach to Glycomics

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and ...

Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While metal-based nanoparticles are associated with synthetic and environmental hassles, cellulose introduces a green, sustainable alternative for nanoparticle synthesis. Here, we present the chemical synthesis and separation procedures to produce new classes of hairy nanoparticles (bearing both amorphous and crystalline regions) and biopolymers based on wood fibers. Through periodate oxidation of soft wood pulp, the glucose ring of cellulose is opened at the C2-C3 bond to form 2,3-dialdehyde groups. Further heating of the partially oxidized fibers (e.g., T = 80 &#176;C) results in three products, namely fibrous oxidized cellulose, sterically stabilized nanocrystalline cellulose (SNCC), and dissolved dialdehyde modified cellulose (DAMC), which are well separated by intermittent centrifugation and co-solvent addition. The partially oxidized fibers (without heating) were used as a highly reactive intermediate to react with chlorite for converting almost all aldehyde to carboxyl groups. Co-solvent precipitation and centrifugation resulted in electrosterically stabilized nanocrystalline cellulose (ENCC) and dicarboxylated cellulose (DCC). The aldehyde content of SNCC and consequently surface charge of ENCC (carboxyl content) were precisely controlled by controlling the periodate oxidation reaction time, resulting in highly stable nanoparticles bearing more than 7 mmol functional groups per gram of nanoparticles (e.g., as compared to conventional NCC bearing &#60;&#60; 1 mmol functional group/g). Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) attested to the rod-like morphology. Conductometric titration, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), electrokinetic-sonic-amplitude (ESA) and acoustic attenuation spectroscopy shed light on the superior properties of these nanomaterials.

Synthesis schemes to prepare highly stable wood fiber-based hairy nanoparticles and functional cellulose-based biopolymers have been detailed.

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Low-energy Cathodoluminescence for (Oxy)Nitride Phosphors

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and flexibility of their emission properties can be achieved by controlling their composition and dopants. However, a lot of work is still required to improve their properties and to reduce the production cost. A possible approach is to correlate the luminescence properties of the Sialon particles with their local structural and chemical environment in order to optimize their growth parameters and find novel phosphors. For such a purpose, the low-voltage cathodoluminescence (CL) microscopy is a powerful technique. The use of electron as an excitation source allows detecting most of the luminescence centers, revealing their luminescence distribution spatially and in depth, directly comparing CL results with the other electron-based techniques, and investigating the stability of their luminescence properties under stress. Such advantages for phosphors characterization will be highlighted through examples of investigation on several Sialon phosphors by low-energy CL.

Low-energy Cathodoluminescence for OxyNitride Phosphors

An excellent chemical and luminescence stabilities of (oxy)nitride phosphors present it as an promising alternative to currently used sulfide and oxide phosphors. In this paper, we present the way to investigate its local luminescence properties using low-energy cathodoluminescence (CL).

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and ...

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

An Aptamer-based Sensor for Unchelated Gadolinium(III)

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples of gadolinium-based contrast agents as a result of incomplete reactions between the ligand and the ion, or as a dissociation product. Since the ion is toxic, its detection is of critical importance. Herein, the design and usage of an aptamer-based sensor (Gd-sensor) for Gd3+ are described. The sensor produces a fluorescence change in response to increasing concentrations of the ion, and has a limit of detection in the nanomolar range (~100 nM with a signal-to-noise ratio of 3). The assay may be run in an aqueous buffer at ambient pH (~7 - 7.4) in a 384-well microplate. The sensor is relatively unreactive toward other physiologically relevant metal ions such as sodium, potassium, and calcium ions, although it is not specific for Gd3+ over other trivalent lanthanides such as europium(III) and terbium(III). Nevertheless, the lanthanides are not commonly found in contrast agents or the biological systems, and the sensor may therefore be used to selectively determine unchelated Gd3+ in aqueous conditions.

An Aptamer-based Sensor for Unchelated GadoliniumIII

The use of polydeoxynucleotide (44-mer aptamer) molecules for sensing unchelated gadolinium(III) ion in an aqueous solution is described. The presence of the ion is detected via an increase in the fluorescence emission of the sensor.

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples ...

An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control cellular functions. The reversal of DNA methylation, or DNA demethylation, is mediated by the ten-eleven translocation (TET) protein family of dioxygenases. Although it has been widely reported that aberrant DNA methylation and demethylation are associated with developmental defects and cancer, how these epigenetic changes directly contribute to the subsequent alteration in gene expression or disease progression remains unclear, largely owing to the lack of reliable tools to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution. To overcome this hurdle, we designed a split-TET2 enzyme to enable temporal control of 5-methylcytosine (5mC) oxidation and subsequent remodeling of epigenetic states in mammalian cells by simply adding chemicals. Here, we describe methods for introducing a chemical-inducible epigenome remodeling tool (CiDER), based on an engineered split-TET2 enzyme, into mammalian cells and quantifying the chemical inducible production of 5-hydroxymethylcytosine (5hmC) with immunostaining, flow cytometry or a dot-blot assay. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype&#8722;phenotype relations in various biological systems.

Detailed protocols for introducing an engineered split-TET2 enzyme (CiDER) into mammalian cells for chemical inducible DNA hydroxymethylation and epigenetic remodeling are presented.

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Iron Nanowire Fabrication by Nano-Porous Anodized Aluminum and its Characterization

Magnetic nanowires possess unique properties that have attracted the interest of different fields of research, including basic physics, biomedicine, and data storage. We demonstrate a fabrication method for iron (Fe) nanowires&#160;via electrochemical deposition into anodic&#160;alumina oxide (AAO) templates. The templates are fabricated by anodization of aluminum (Al) discs, and the pore length and diameter are controlled by changing the anodizing conditions. Pores with an average diameter of around 120 nm are created using oxalic acid as the electrolyte. Using this method, cylindrical nanowires are synthesized, which are released by dissolving the alumina using a selective chemical etchant.

In this work, we describe a&#160;protocol to fabricate iron nanowires, including the formation of the porous alumina membrane that is used as the template, electrodeposition into templates using electrolyte solution, and the release of the nanowires into the solution.

Magnetic nanowires possess unique properties that have attracted the interest of different fields of research, including basic physics, biomedicine, and ...

Organic Structure-directing Agent-free Synthesis for *BEA-type Zeolite Membrane

Membrane separation has drawn attention as a novel-energy saving separation process. Zeolite membranes have great potential for hydrocarbon separation in petroleum and petrochemical fields because of their high thermal, chemical, and mechanical strength. A *BEA-type zeolite is an interesting membrane material because of its large pore size and wide Si/Al range. This manuscript presents a protocol for *BEA membrane preparation by a secondary growth method that does not use an organic structure-directing agent (OSDA). The preparation protocol consists of four steps: pretreatment of support, seed preparation, dip-coating, and membrane crystallization. First, the *BEA seed crystal is prepared by conventional hydrothermal synthesis using OSDA. The synthesized seed crystal is loaded on the outer surface of a 3 cm long tubular &#945;-Al2O3 support by a dip-coating method. The loaded seed layer is prepared with the secondary growth method using a hydrothermal treatment at 393 K for 7 days without using OSDA. A *BEA membrane having very few defects is successfully obtained. The seed preparation and dip-coating steps strongly affect the membrane quality.

A *BEA seed crystal was loaded on a porous &#945;-Al2O3 support by the dip-coating method, and hydrothermally grown without using an organic structure-directing agent. A *BEA-type zeolite membrane having very few defects was successfully prepared by the secondary growth method.

Membrane separation has drawn attention as a novel-energy saving separation process. Zeolite membranes have great potential for hydrocarbon separation in ...