Anticancer Camptothecin- N-Poly(lactic acid) Nanoconjugates with Facile Hydrolysable Linker

Abstract

We report a strategy of conjugating CPT to the terminal carboxylate group of polylactide (PLA) with a facile hydrolysable amino ester linker via a controlled polymerization method. The obtained CPT-N-PLA conjugates were able to self-assemble into 50-100 nanometer-sized conjugates (NCs) with desired in vitro physicochemical properties and showed enhanced in vivo therapeutic efficacy against Lewis lung carcinoma (LLC) induced in C57BL/6 mice.

Figure 1

Preparation of PEGylated CPT-N-PLA NCs via CPT-N-OH initiated LA polymerization in the presence of (BDI-_EI)ZnN(TMS)2, followed by nanoprecipitation and non-covalent surface modification.

Figure 2

(A) Overlay of GPC traces of CPT-N-PLA_n (n = 50, 100 and 200). (B) (BDI-EI)ZnN(TMS)₂/CPT mediated controlled ring-opening polymerization (ROP) of LA at various ratio of LA/CPT (MWD = Molecular Weight Distribution. (C) HPLC analysis of CPT-N-OH initiated polymerization and release of CPT from CPT-N-PLA_n NC. (HPLC traces from top to bottom: free CPT, CPT-N-OH, CPT-N-PLA, CPT released from CPT-N-PLA NC).

Figure 3

Formulation and characterization of CPT-N-PLA₁₀ NCs. (A) DLS analysis of CPT-N-PLA₁₀ NC in water (0.5 mg/mL). (B) Precipitation of CPT-N-PLA₁₀ from DMF solution into water at various CPT-N-PLA₁₀ concentrations. (C) Stability of PEGylated CPT-N-PLA₁₀ NCs in PBS, cell culture medium, and human serum buffer (human serum:PBS=1:1, v/v). (D) DLS analysis of NCs reconstitution: Monodispersed PEGylated CPT-N-PLA₁₀ NCs mixed with bovine serum albumin (BSA) in water before lyophilization (upper panel). The reconstituted NC after lyophilization in the presence of BSA (BSA:NC=10:1, wt/wt) (bottom panel).

Figure 4

(A) Release kinetics of CPT from PEGylated CPT-PLA₁₀ NC and PEGylated CPT-N-PLA₁₀ NC in human serum buffer (human serum:PBS=1:1, v/v) at 37 °C. (B) Cytotoxicity of free CPT, PEGylated CPT-_LA10 NC and PEGylated CPT-N-PLA₁₀ in MCF-7 cells as determined by MTT assay (37 °C, 72 h). Statistical differences between the groups were assessed with Student’s t-test. * P< 0.05 is considered statistically significant.

Figure 5

In vivo tumor reduction study. (A) Experimental procedures of the study. (B) Delay and inhibition of LLC (Lewis lung carcinoma) tumor growth in C57BL/6 mice with different treatments (PEGylated CPT-LA₁₀ NC, PEGylated CPT-N-LA₁₀ NC, irinotecan, mPEG-PLA NC, and PBS), N=6. Data are presented as relative median tumor size (V/V₀, compare to the tumor volume at day 0). (C) Box plot of LLC tumor growth in C57BL/6 mice at day 8, after treated with PEGylated CPT-LA NC, PEGylated CPT-N-LA NC, irinotecan, mPEG-PLA NC, and PBS at day 0. Statistical properties of relative tumor volume ratio (V/V₀, compare to the tumor volume at day 0) shown in box plot are as follows: box (median with 25/75% percentile), whisker (5/95% percentile), and asterisks (maximum/minimum). Univariate differences between the groups were assessed with Mann-Whitney U test. P< 0.05 is considered statistically significant. (D) Relative body weight (M/M₀) monitoring over the study (M: body weight monitored during the study; M₀: body weight monitored at day 0). (E) Representative deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end (TUNEL) staining sections of LLC tumors with all treatments (PEGylated CPT-LA₁₀ NC, PEGylated CPT-N-LA₁₀ NC, irinotecan, mPEG-PLA NC, and PBS). Scale bar: 40 μm.