Controlled synthesis of camptothecin-polylactide conjugates and nanoconjugates

Abstract

We report here a unique method of formulating camptothecin-polylactide (CPT-PLA) conjugate nanoparticles, termed nanoconjugates (NCs), through CPT/(BDI)ZnN(TMS)(2) [(BDI) = 2-((2,6-diisopropylphenyl)amido)-4-((2,6-bisalkyl)-imino)-2-pentene] mediated polymerization of lactide (LA) followed by nanoprecipitation. When CPT was used as the initiator to polymerize LA in the presence of (BDI)ZnN(TMS)(2), the polymerization was completed within hours with nearly 100% CPT loading efficiency and 100% LA conversion. CPT loading as high as 19.5% can be achieved for the CPT-polylactide (CPT-PLA) conjugate prepared at a LA/CPT ratio of 10. The steric bulk of the chelating ligands and the type of metals used had a dramatic effect on the initiation of the LA polymerization and the tendency of the ring-opening of the CPT lactone. The CPT/(BDI)ZnN(TMS)(2)-mediated LA polymerization yielded CPT-PLA conjugates with well-controlled molecular weights and narrow molecular weight distributions (1.02-1.18). The nanoprecipitation of CPT-PLA led to the formation of NCs around 100 nm in size with narrow particle size distributions. Sustained release of CPT from CPT-PLA NCs was achieved without burst release. CPT-PLA NCs were toxic to PC-3 cells with tunable IC(50) possible by adjusting the drug loading of the CPT-PLA NCs.

Figure 1

(a) Schematic illustration of (BDI-3)ZnN(TMS)₂/CPT mediated ring-opening polymerization of LA to make CPT-PLA conjugates followed by nanoprecipitation of the resulting CPT-PLA conjugates for the preparation of CPT-PLA nanoconjugates; (b) The structure of (BDI-m)ZnN(TMS)₂ (m = 1–4).

Figure 2

(a) (BDI-3)ZnN(TMS)₂/CPT mediated ring-opening polymerization of LA at various LA/CPT ratios; (b) Overlay of the GPC trace of CPT-LA₇₅, CPT-LA₂₀₀ and CPT-LA₄₀₀ prepared by (BDI-3)ZnN(TMS)₂/CPT mediated LA polymerizations.

Figure 3

(a) HPLC analysis of the reaction of SA and CPT in the presence of (i) TEA, (ii) (BDI-1)MgNTMS₂, (iii) (BDI-1)ZnNTMS₂, (iv) (BDI-3)ZnNTMS₂ or (v) (BDI-4)ZnNTMS₂; (b) Structure and H-assignment of CPT-SA. (c) ESI-MS of CPT-SA (positive mode). [M+H]⁺: m/z 449.1. High resolution ESI-MS for [M+H]⁺ obtained: m/z 449.1355; calculated: m/z 449.1349; (d) ¹H NMR of CPT-SA (CD₃OD, 500 MHz). (e) The chemical shift values of CPT and CPT-SA derived from the corresponding ¹H-NMR spectra

Figure 4

(a) Analysis of the particle size and size distribution of CPT-LA₂₅ NC by DLS. (b) Scanning electron microscope (SEM) image of CPT-LA₂₅ NC. Scale bar = 1.0 µm

Figure 5

(a) Release of CPT from the CPT-LA₁₀ NC (■) and the CPT/PLA NP prepared by encapsulation (▲, CPT/PLA = 5/95 (wt/wt)); (b) MTT assay to evaluate the cytotoxicity of CPT-LA₁₀, CPT-LA₂₅, CPT-LA₅₀ NCs and CPT in PC-3 cell (37°C, 72 h).

Figure 6

(a) Overlay of the HPLC spectrum of (a) authentic CPT and (b) CPT released from CPT-LA₂₅ NC (incubated in PBS for 2 days at 37°C. (b) ¹H NMR spectrum (DMSO-d⁶) of the CPT released from CPT-LA₂₅ and collected on a preparative RP HPLC, and compared with the ¹H NMR spectrum of the authentic CPT

Scheme 1

Equilibrium of CPT Lactone and Carboxylate Forms

Scheme 2

Suggested Insertion–Coordination Mechanism of (BDI)Zn-OR Mediated Ring Opening of (a) LA and (b) SA.