News Column

"Caged Platinum Nanoclusters for Anticancer Chemotherapeutics" in Patent Application Approval Process

September 12, 2014



By a News Reporter-Staff News Editor at Drug Week -- A patent application by the inventor LIN, Shu-Yi (Miaoli County, TW), filed on February 18, 2014, was made available online on August 28, 2014, according to news reporting originating from Washington, D.C., by NewsRx correspondents (see also National Health Research Institutes).

This patent application is assigned to National Health Research Institutes.

The following quote was obtained by the news editors from the background information supplied by the inventors: "Unlike the toxicity of cisplatin, which is activated simply by water. Pt is considered to he a noble metal that can only dissolve in highly corrosive agents such as aqua regia (HNO.sub.3/HCl), which initially oxidizes and then dissolves Pt to form Pt chloride complexes. It has been recently discovered that the degree of oxidization can he increased significantly by decreasing the Pt size to increase the surface-to-volume ratio to allow oxygen adsorption and facilitate water oxidation for surface corrosion. However, finding a method to shrink Pt efficiently remains a challenge."

In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventor's summary information for this patent application: "In one aspect, the invention relates to a double-caged platinum nanocluster complex, comprising: (a) a dendrimer; (b) a platinum nanocluster comprising platinum oxides, the platinum nanocluster being confined inside of the dendrimer; and polyethylene glycol (PEG), coated on the surface of the dendrimer. The dendrimer may be an amine-terminated dendrimer.

"In another aspect, the invention relates to a caged platinum nanocluster complex, comprising: (a) an amine-terminated dendrimer; and (b) a platinum nanocluster comprising platinum oxides and having an average diameter of 0.93 nm with a standard deviation of 0.22 nm, the platinum nanocluster being confined inside of the amine-terminated dendrimer.

"Further in another aspect, the invention relates to a method for synthesizing a caged platinum nanocluster complex as aforementioned, comprising the steps of: (a) admixing a first solution comprising octahedronal hexachloroplatinate anions with a second solution comprising an amine-terminated dendrimer or a hydroxyl-terminated dendrimer to form a mixture comprising a PtCl.sub.6.sup.2- anion/dendrimer complex; (b incubating the mixture comprising the PtCl.sub.6.sup.2- anion/dendrimer complex for a sufficient period; reducing the PtCl.sub.6.sup.2- anion in the PtCl.sub.6.sup.2- anion/dendrimer complex to form a mixture comprising a dendrimer caged platinum nanocluster complex; (d) passing the mixture comprising the dendrimer caged platinum nanocluster complex through a filter to obtain a filtrate comprising the dendrimer caged platinum nanocluster complex; and (e) freeze-drying the filtrate to obtain the dendrimer caged platinum nanocluster complex.

"Further in another aspect, the invention relates to a method for synthesizing a double caged platinum nanocluster complex comprising: (i) dissolving a dendrimer caged platinum nanocluster complex as aforementioned in a solvent to form a solution, the complex comprising: (a) an amine-terminated dendrimer or a hydroxyl-terminated dendrimer; and (b) a platinum nanocluster comprising platinum oxides, being confined inside of the amine-terminated or the hydroxyl-terminated dendrimer; (ii) adding PEG-aldehyde into the solution provided that the dendrimer is amine-terminated or adding PEG-NH.sub.2 into the solution provided that the dendrimer is hydroxyl-terminated; and (iii) allowing the PEG-aldehyde to react with primary amines of the amine-terminated dendrimer, or allowing the PEG-NH.sub.2 to react with the hydroxyl-terminated dendrimer, and thereby obtaining the double caged platinum nanocluster complex.

"Further in another aspect, the invention relates to a method of suppressing tumor cell growth, comprising administering to a subject in need thereof an effective amount of the complex as aforementioned.

"Yet in another aspect, the invention relates to a pharmaceutical composition comprising: (a) a therapeutically effective amount of a complex as aforementioned; and (b) a pharmaceutically acceptable carrier.

"These and other aspects will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.

"The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

"FIG. 1A is a schematic representation of a novel strategy based on tuning anionic geometry for the formation of PN; paths (i) and (ii) show that the outward and inward of G.sub.2NH.sub.2 can associate selectively with square planar PtCl.sub.4.sup.2- and octahedronal PtCl.sub.6.sup.2-, respectively.

"FIG. 1B is a schematic representation of the caged PN mixed with a tumor-penetrating peptide to target the tumor and kill malignant cells by shedding, the outer PEG corona to exert tumor-inside activation. The key point of anticancer efficiency was based on the PN losing its intrinsic inertness (inset of red rectangle) to exhibit its dissolution in weakly acidic organelles.

"FIGS. 2A-B show Raman spectra of the PtCl.sub.4.sup.2-/G.sub.2NH.sub.2 complex (A) and PtCl.sub.6.sup.2-/G.sub.2NH.sub.2 complex (B), showing the N--Pt--N coordination from the former complex.

"FIGS. 2C-D show HRTEM images of the PtCl.sub.4.sup.2-/G.sub.2NH.sub.2 complex (C) and the PtCl.sub.6.sup.2-/G.sub.2NH.sub.2 complex (D) before reduction.

"FIGS. 2E-F show that while the complex of (C) and (D) were reduced, the PN was caged in the G.sub.2NH.sub.2 exterior (E) and interior (F). All TEM samples were prepared fresh before two days of the measurement, and the as-prepared samples were naturally dried. All measurements were taken in the absence of negative staining.

"FIG. 3A show that the IC.sub.50 of CPN was higher than the IC.sub.50 of both cisplatin and carboplatin. The magnified inset shows the lower concentration, ranging from 0 to 50 .mu.g/mL

"FIG. 3B are microscope images for observing the pathway of cell death. MDA-MB-231 cells that were untreated or treated with CPN at approximately 50 .mu.g/ml, for different times were double-stained with annexin V-FITC and PI. The upper panels from left to right show untreated cells and cells treated with CPN for 5 h. respectively. The lower panels show the cells treated with CPN for 10 h and 24 h, respectively. Scale bar: 30 .mu.m.

"FIGS. 4A-B are XPS spectra showing the binding energy of Pt (A) and oxygen (B), specially represented in (A) was the Pt(4f.sub.7/2) and Pt(4f.sub.5/2) region of the CPN and Pt nanoparticles. The asterisk indicates that the binding energy of the CPN had shifted to 73.4 eV. The binding energy was calibrated by the An 4f.sub.7/2(83.7 eV) peak of gold film deposited on the substrate as an internal standard.

"FIG. 4C shows various endocytosis inhibitors used during cellular uptake to verify the internalization pathway of the CPN.

"FIG. 4D shows confocal microscopy of the cellular uptake behavior of CPN on MDA-MB-23I cells; the CPN was labeled a FITC for visualization (green) and lysosomal tracker (red), and the co-localization of CPN and lysosomal tracker was yellow color. The cell morphology has changed due to the CPN toxicity.

"FIG. 4E shows the dissolution of Pt from CPN of the invention to ionic form under an endosomal-mimicking environment as determined by ICP-MS. The CPN was dissolved in aqua regia (3:1 HCl/HNO.sub.3) as a control.

"FIGS. 5A-F show anticancer therapeutic effects of CPN (A-C) and DCPN (D-F) evaluated after IT and IV injection, respectively. A) and E) Serial tumor volume, B) Representative images of tumor size at the end point for each group, C) the apoptotic cell death was observed using a TUNEL assay (scale bar: 20 .mu.m), D) tumor target, and F) body weight were recorded. The sizes of nude mice bearing tumors were reached approximately 60 mm.sup.3 and 150 mm.sup.3 for IT and IV injection, respectively. The animal models were treated with two sets of treatments, which included IT injection (PBS, G.sub.2NH.sub.2, the CPN and cisplatin) and IV injection (PBS, G.sub.2NH.sub.2, iRGD, the DCPN and cisplatin). The profiles of G.sub.2NH.sub.2 in IT and iRGD alone in IV were omitted to avoid a complicated graph. The tumor volumes and body weight of mice were measured on the indicated days. Each data point represents the relative changes in mean tumor volume (n=5, P

"FIG. 6 shows an EDS spectrum of the CPN. The asterisks represent Pt signals.

"FIGS. 7A-C show comparisons of the properties of CPN and PN assembly. These materials were produced by tuning anionic geometry. A) Solubility representation. B) Cytotoxicity evaluation. C) intracellular uptake efficacy.

"FIGS. 8A-D are microscopic images to compare cisplatin-induced cell death and CPN-induced cell death pathways. MDA-MB-231 cells that were untreated or treated with 30 .mu.g/mL cisplatin for different times were double-stained with annexin V-FITC and PI. The upper panels from left to right show untreated cells (A) and cells treated (B) with cisplatin for 5 h, respectively. The lower panels show the cells treated with CPN for 10 h (C) and 24 h (D), respectively. Scar bar: 50 .mu.m.

"FIG. 9 shows the ROS production of the CPN. MDA-MB-231 cell line was cultured in RPMI 1640 medium (media were supplemented with 10% fetal bovine serum) under 5% CO.sub.2 atmosphere at 37.degree. C. The cells were inoculated with 1.times.10.sup.5 cells per well in a 6-well cell-culture plate and cultivated at 37.degree. C. for 24 h. Then, the culture medium was changed with the presence of various treatments, including G.sub.2NH.sub.2 alone, the CPN, cisplatin, and 0.03% H.sub.2O.sub.2 in the culture medium. The tests were performed for 3 h without changing the medium. After removal of the supernatant of cell culture medium, the cells were isolated for ROS assay using CM-H.sub.2DCFDA from INVITROGEN.TM.

"FIG. 10A shows the estimated safe dosage and treatment time of BA. It is noteworthy that the BA is also well-known to easily cause MDA-MB-231 cell death, through impairing mitochondrial functions. Thus, the concentration of BA and treatment time should he minimized and shortened, respectively, avoiding cell death from the synergistic effect of inhibitor and CPN, resulting in no significant difference between the presence and absence of BA during the CPN treatment.

"FIG. 10B shows endosomal/lysosomal pH-rising effects on the CPN cytotoxicity.

"FIG. 11 shows apoptotic cell death induced by CPN observed by active caspase-3 immunostaining. Scale bar: 20 .mu.m.

"FIGS. 12A-C show .sup.3H NMR spectra from DCPN and its precursors.

"FIG. 13 shows bio-clearance of CPN after the end point.

"FIG. 14 shows comparative efficiency in tumor-targeting and anticancer chemotherapeutics of the G.sub.2NH.sub.2 coated with a cleavable PEG. No significant efficacy in tumor suppression was detected. The tumor mass at the end point is significantly larger than the one at the initial point.

"FIG. 15 shows feed intake of mice as recorded. Each data point represents the total amount of feed intake in each cage (n=5) after drug injection.

"FIGS. 16A-D show comparative histological H&E staining in the excised tumors. Images A)-D), various treatments including PBS, G.sub.2NH.sub.2, DCPN, and cisplatin at 400.times. magnification."

URL and more information on this patent application, see: LIN, Shu-Yi. Caged Platinum Nanoclusters for Anticancer Chemotherapeutics. Filed February 18, 2014 and posted August 28, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=3282&p=66&f=G&l=50&d=PG01&S1=20140821.PD.&OS=PD/20140821&RS=PD/20140821

Keywords for this news article include: Alkylating Agents, Antineoplastics, Chlorine Compounds, Cisplatin, Drugs, National Health Research Institutes, Nitrogen Compounds, Pharmaceuticals, Platinum Compounds, Therapy.

Our reports deliver fact-based news of research and discoveries from around the world. Copyright 2014, NewsRx LLC


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Source: Drug Week


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