Functionalized Block Co‐Polymer Pro‐Drug Nanoparticles with Anti‐Cancer Efficacy in 3D Spheroids and in an Orthotopic Triple Negative Breast Cancer Model

Abstract

Amphiphilic block co-polymers composed of poly(ethylene glycol)-co-poly(lactide)-co-poly(2-((tert-butoxycarbonyl)amino)-3-propyl carbonate) (PEG-pLA-pTBPC) are synthesized in monomer ratios and arrangements to enable assembly into nanoparticles with different sizes and architectures. These materials are based on components in clinical use, or known to be biodegradable, and retain the same fundamental chemistry across "AB" and "BAB" block architectures. In MCF7 and MDA-MB-231 breast cancer cells, nanoparticles of <100 nm are internalized most rapidly, by both clathrin-and caveolin-mediated pathways. In THP-1 cells, polymer architecture and length of the hydrophilic block is the most important factor in the rate of internalization. The organ distributions of systemically injected nanoparticles in healthy mice indicate highest accumulation of the BAB-blocks in lungs and liver and the lowest accumulation in these organs of a methoxyPEG 5000-pLA-pTBPC polymer. Conjugation of doxorubicin via a serum-stable urea linker to the carbonate regions of PEG 5000-pLA-pTBPC generates self-assembling nanoparticles which are more cytotoxic in 2D, and penetrate further in 3D spheroids of triple negative breast cancer cells, than the free drug. In an aggressive orthotopic triple negative breast cancer mouse model, the methoxyPEG 5000-pLA-pTBPC is of similar potency to free doxorubicin but with no evidence of adverse effects in terms of body weight.

Polymer Pro-Drug Nanoparticles for Sustained Release of Cytotoxic Drugs Evaluated in Patient-Derived Glioblastoma Cell Lines and In Situ Gelling Formulations

“…Following a 4 h incubation with NPs, co-localisation of NP-Cy5 (red) and Lysotracker green (green) was observed in GIN8, GIN28 and U87 cells, indicating the internalisation of NP-Cy5 P2 was occurring via endocytosis and trafficked to lysosomes. Such intracellular trafficking following endocytosis has been widely reported for polymeric NPs [35,36] and is in agreement with previous studies on our NPs in different cell lines [16,37]. As apparent in Figure 7, a burst release of DOX from the NPs occurred over the first 6 h ( Figure 7B(B I )), with over 60% of DOX released within the first 24 h. After this time point, steady state release was observed, with just over 80% of the drug cumulatively released over the following 44 days ( Figure 7B).…”

“…Furthermore, a burst release of the drug from the P6 DOX-NPs in the microparticulate paste matrix (mimicking the intended surgical application) was observed during the first 8 h, whereas a slower release occurred from the moulds. In both the paste and mould formulations a sustained release profile was observed after 24 h and up to 2 months, and for the paste ~70% of DOX was released after While the decrease in drug potency is not unexpected over the time periods of 2D cell culture assays, as internalisation of free drug is more rapid than endocytosis and pro-drug activation from a polymer chain, in previous studies these polymer DOX-NPs achieved greater than or equal potency to free DOX in breast, lung, skin and intestinal tumour cells [16,37]. We suggest therefore that the specific GBM cells studied, either internalised the NPs less efficiently than the free drug over the time periods of the assay and/or that the urea linker in the DOX polymer conjugates was more stable in these cell lines than in those assayed previously.…”

“…The polymers used in this study are shown in Scheme 1 and their key attributes in Table 1. The polymers were all synthesised by ring-opening polymerisation, as described previously [16,17], and were characterised by NMR spectroscopy and gel permeation chromatography (GPC), as shown in Table 1. The polymers were all synthesised by ring-opening polymerisation, as described previously [16,17], and were characterised by NMR spectroscopy and gel permeation chromatography (GPC), as shown in Table 1.…”

“…Doxorubicin (hydrochloride) (DOX.HCl) was purchased from Cayman Chemical company (Ann Arbor, MI, USA). Synthesis of all the polymers was via ring opening of lactide and a t-butyloxycarbamoyl-protected cyclic carbonate monomer (TBPC) derived from serinol by methoxypoly(ethyleneglycol)s to generate polymers abbreviated as mPEG 5000 -(LA) n -(TBPC) y , followed by Cy5-labelling and DOX coupling to produce the final pro-drug platforms were performed following previously reported protocols [ 16 , 17 ].…”

“…We have recently reported polymer pro-drugs based on a PEGylated poly(lactide)-poly(carbonate) copolymer (mPEG n -LA m -TBPC p ) [ 16 , 17 ], which is based on well-established chemistries and components already in clinical use. The polymer contains a carbamate moiety, in the side-chains of the monomer units, which after simple deprotection chemistry generates a free primary amine functionality, offering a simple conjugation point for drugs to be linked to the polymer backbone [ 16 , 17 ]. The polymer contains primary amine functionality in the side-chains of one of the monomer units, offering a simple conjugation point for drugs to be linked to the polymer backbone.…”