Polymeric Micelles with Pendant Dicarboxylato Chelating Ligands Prepared via a Michael Addition for <i>cis</i>-Platinum Drug Delivery

A new monomer with a neighboring carboxylate functional group was prepared via carbon Michael addition between ethylene glycol dimethacrylate and malonate. The monomer, 1,1-di-tert-butyl 3-(2-(methacryloyloxy)ethyl) butane-1,1,3-tricarboxylate (MAETC), was polymerized in a controlled manner using RAFT polymerization. After deprotection and the conjugation of platinum drugs, a macromolecular Pt complex was created, which was found to be insoluble in water. 195Pt NMR revealed that the desired complex has been formed next to a minor fraction of other Pt complexes. Block copolymers were prepared using poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMEMA) as macroRAFT agent for chain extension with the synthesized monomer to yield three different block copolymers with varying PMAETC block lengths. Subsequent conjugation to platinum resulted in amphiphilic block copolymers, which can ultimately generate micelles. The length of the core block had significant contribution to the micelle sizes with the micelle size increasing with an increase of the hydrophobic block length. The polymers prior to platinum conjugation were found to be nontoxic when in contact with A549, a lung cancer cell line. After conjugation with the platinum drug, the micelle with the shortest PMAETC block length was found to have the highest toxicity, which may be due to the fastest cisplatin release when compared to the longer PMAETC block lengths

Core-Cross-Linked Micelles Synthesized by Clicking Bifunctional Pt(IV) Anticancer Drugs to Isocyanates

Most low molecular weight platinum-based anticancer drugs have a short circulation time in the bloodstream. One of the potential strategies to improve the targeted delivery of cisplatin and prolong its circulation is via the use of nanocarriers. An improved drug delivery system was developed via reversible addition−fragmentation chain transfer (RAFT) polymerization. In a one-pot reaction, the incorporation of anticancer drug and core cross-linking was simultaneously carried out by using the highly effective reaction of isocyanate groups in the core of the polymeric micelles poly(oligo(ethylene glycol) methyl ether methacrylate)-block-poly(styrene-co-3-isopropenyl-α,α-dimethylbenzyl isocyanate) (POEGMA-block-P(STY-co-TMI)) with amine groups in the prepared platinum(IV) drug. The micelles with platinum(IV) incorporated with a size of 36 nm were very stable in water. In a reductive environment, in this study simulated using ascorbate, the drug was released at a slow rate of 82% in 22 days and at the same time the cross-linked micelle broke down into free block copolymers as evidenced using inductively coupled plasma-mass spectrometer (ICP-MS), size exclusion chromatography (SEC), and dynamic light scattering (DLS). The in vitro study also revealed the promising antitumor activity of prepared platinum(IV) drugs encapsulated into the micelle structure

Thiol–yne and Thiol–ene “Click” Chemistry as a Tool for a Variety of Platinum Drug Delivery Carriers, from Statistical Copolymers to Crosslinked Micelles

Statistical and block copolymers based on poly(2-hydroxyethyl methacrylate) (PHEMA) and poly[oligo(ethylene glycol) methylether methacrylate] (POEGMEMA) were modified with 4-pentenoic anhydride or 4-oxo-4-(prop-2-ynyloxy)butanoic anhydride to generate polymers with pendant vinyl or acetylene, respectively. Subsequent thiol–ene or thiol–yne reaction with thioglycolic acid or 2-mercaptosuccinic acid leads to polymers with carboxylate functionalities, which were conjugated with cisplatin (cis-diamminedichloroplatinum(II) (CDDP)) to generate a drug carrier for Pt-drugs. Only the polymers modified with 2-mercaptosuccinic acid resulted in the formation of soluble well-defined polymers with gel formation being prevented. Due to the hydrophobicity of the drug, the block copolymers took on amphiphilic character leading to micelle formation. The micelles were in addition crosslinked to further stabilize their structure. Pt-containing statistical copolymer, micelles, and crosslinked micelles were then tested regarding their cellular uptake by the A549 lung cancer cell line to show a superior uptake of crosslinked micelles. However, due to the better Pt release of the statistical copolymer, the highest cytotoxicity was observed with this type of polymer architecture

Correlation of Asian and Western populations with variables of interest, using Fisher’s exact test.

a–f<p>Analyses excluding missing data in ^agrade: Western n = 9, Asian n = 2, ^breceptor: Western n = 13, Asian n = 3, ^cradiotherapy: Western n = 18, Asian n = 3, ^dchemotherapy: Western n = 19, Asian n = 4, ^esurgery: Western n = 24, Asian n = 6, ^fendocrine therapy: Western n = 18, Asian n = 7.</p

Multivariate analysis for disease-free survival in the hormone receptor positive cohort.

<p>Multivariate analysis for disease-free survival in the hormone receptor positive cohort.</p

Univariate analysis for disease-free survival.

<p>Univariate analysis for disease-free survival.</p

Baseline patient demographics.

a<p>The percentages may not add up to one hundred percent due to rounding.</p

Immunomagnetic melanoma cell isolation.

The indicated melanoma cells (n = 100) were spiked into pre-enriched PBMCs in triplicate experiments and then recovered using immunomagnetic beads (Rare Cell Kit, Fluxion) coupled to either (A) anti human MCAM antibody clone H1P12 or F4-35H7 or (B) to anti human MCAM antibody clone H1P12 (αMCAM) or anti human MCSP antibody clone 9.2.27 (αMCSP) or with the combination of both. The proportion of isolated melanoma cells is presented. (C) 14 advanced melanoma patients (16 blood draws, 3x9ml each) were compared for CTC counts after αMCAM (H1P12) based, αMCSP (9.2.27) based or combination based CTC isolation. CTC counts are graphed as box blot.</p

Melanoma CTC counts and PD-L1 positive CTCs.

Melanoma CTC counts and PD-L1 positive CTCs.</p

PD-L1 on melanoma CTCs.

(A) Representative melanoma CTC identification staining with PD-L1 detection of a patient derived CTC surrounded by lymphocytes. (B) MelRM, NM176 or SKMel28 cells were spiked into blood samples of healthy donors and isolated immediately or after room temperature blood storage for the indicated time before recovery using our melanoma CTC isolation and immunostaining protocol including PD-L1 probing. Data from three to six experiments (different healthy donors) were analysed per cell line. Mel-ID: probed with cocktail of three fluorescently conjugated melanoma identification antibodies.</p