Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers - PubMed (original) (raw)

Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers

Frank Gu et al. Proc Natl Acad Sci U S A. 2008.

Abstract

There has been progressively heightened interest in the development of targeted nanoparticles (NPs) for differential delivery and controlled release of drugs. Despite nearly three decades of research, approaches to reproducibly formulate targeted NPs with the optimal biophysicochemical properties have remained elusive. A central challenge has been defining the optimal interplay of parameters that confer molecular targeting, immune evasion, and drug release to overcome the physiological barriers in vivo. Here, we report a strategy for narrowly changing the biophysicochemical properties of NPs in a reproducible manner, thereby enabling systematic screening of optimally formulated drug-encapsulated targeted NPs. NPs were formulated by the self-assembly of an amphiphilic triblock copolymer composed of end-to-end linkage of poly(lactic-co-glycolic-acid) (PLGA), polyethyleneglycol (PEG), and the A10 aptamer (Apt), which binds to the prostate-specific membrane antigen (PSMA) on the surface of prostate cancer (PCa) cells, enabling, respectively, controlled drug release, "stealth" properties for immune evasion, and cell-specific targeting. Fine-tuning of NP size and drug release kinetics was further accomplished by controlling the copolymer composition. By using distinct ratios of PLGA-b-PEG-b-Apt triblock copolymer with PLGA-b-PEG diblock copolymer lacking the A10 Apt, we developed a series of targeted NPs with increasing Apt densities that inversely affected the amount of PEG exposure on NP surface and identified the narrow range of Apt density when the NPs were maximally targeted and maximally stealth, resulting in most efficient PCa cell uptake in vitro and in vivo. This approach may contribute to further development of targeted NPs as highly selective and effective therapeutic modalities.

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Conflict of interest statement

Conflict of interest statement: O.C.F. and R.L. have financial interest in BIND Biosciences. The rest of the authors declare no conflict of interest.

Figures

Fig. 1.

Development of PSMA-targeted NPs by using PLGA-_b_-PEG-_b_-Apt TCP. (A) The PLGA-_b_-PEG-_b_-Apt-biointegrated TCP was synthesized in two steps: (i) synthesis of PLGA-_b_-PEG by conjugating carboxyl-capped PLGA (PLGA-acid) to the amine terminals of heterobifunctional PEG (amine-PEG-acid) and (ii) formation of PLGA-_b_-PEG-_b_-Apt by conjugating the carboxyl ends of PLGA-_b_-PEG-acid to the amine ends of A10 PSMA Apt. (B) 1H NMR characterization of PLGA-_b_-PEG and PLGA-_b_-PEG-_b_-Apt. For the synthesis of PLGA-_b_-PEG, the yield of PLGA and PEG conjugation was 73–91% and the purified PLGA-_b_-PEG DCP was used for the subsequent conjugation to Apt. The presence of Apt on the PLGA-_b_-PEG-_b_-Apt TCP was visualized by the peaks between 1.8 and 2.2 ppm. The Apt conjugation efficiency of the PLGA-_b_-PEG DCP for seven independent reactions was 13–21%. (C) By titration in water, the PLGA-_b_-PEG-aptamer TCPs self-assemble and form PSMA-targeted NP-Apt bioconjugates. By using distinct ratios of PLGA-_b_-PEG-_b_-Apt TCP with PLGA-_b_-PEG DCP lacking the A10 Apt during NP formulation, the Apt surface density can be precisely and reproducibly changed.

Fig. 2.

Fine-tuning the nanoparticle physiochemical properties by using PLGA-_b_-PEG-_b_-Apt TCPs. (A) Effect of TCP composition on NP size. Synthesis of TCP containing different molecular mass segments of PLGA and PEG. Targeted NPs were formulated by TCP self-assembly. The size of each NP formulation was measured by dynamic light scattering. Each data point represents the average of four experiments (n = 4). The standard deviation varied between 1 and 6%. The range of nanoparticle size polydispersity index (PDI) was between 0.05 and 0.10 and that the of zeta potential of various nanoparticles formulations was between −20 and −29. (B) NP drug release properties as a function of PLGA-_b_-PEG-_b_-Apt TCP composition. Dtxl was encapsulated into various NPs at a mass loading of 1.5 wt/wt%. Each data point represents the average of four experiments (n = 4). (C) Quantification on the percentage of NP accumulation in LNCaP and PC3 cells. NP-Apt bioconjugates were formulated by self-assembly of the PLGA-_b_-PEG-_b_-Apt TCP containing different lengths of PLGA and PEG segments. LNCaP and PC3 cells were incubated with 50 μg of 3H-labeled PLGA-_b_-PEG-_b_-Apt NPs for 30 min. The amount of NPs endocytosed was determined by 3H radiation counts collected in the cells. Each data point represents the average of four experiments (n = 4).

Fig. 3.

NP-Apt cellular uptake. The NBD dyes (green) were formulated into PLGA-PEG-aptamer triblock nanoparticles by nanoprecipitation. LNCaP (PSMA+) and PC3 (PSMA−) cells were incubated with 50 μg of NBD-encapsulated PLGA-PEG-aptamer nanoparticles for 30 min. The early and late endosomal markers were visualized in red. The cell nuclei were stained by DAPI (blue).

Fig. 4.

Fine-tuning the Apt ligand density for targeted cell uptake in vitro. (A) Estimation of Apt ligand density on NP surface. The aptamer density can be precisely controlled in a linear manner by varying the ratio of PLGA0.67-_b_-PEG3400-_b_-Apt and PLGA0.67-_b_-PEG3400. (B) NPs containing 0.05–10% of PLGA0.67-PEG3400-Apt were synthesized by mixing with different amounts of PLGA0.67-PEG3400. LNCaP and PC3 cells were incubated with 3H-labeled PLGA-PEG-Apt NPs for 30 min (black) and 2 h (white). Data represent the mean and the standard error of mean of triplicates per group. Data points for NP-Apt 0.1–10% were statistically significant compared with NP-Apt 0% by t test at 95% confidence interval. One-way ANOVA with Fisher's least significant difference (LSD) post hoc comparisons at 95% confidence interval was used for statistical comparisons between groups.

Fig. 5.

The effect of Apt surface density on NP biodistribution in vivo. The comparative biodistribution study of a single systemic administration of NPs containing different 1% (green), 5% (blue), and 10% (cyan) of NP-Apt were administered by retro-orbital injection into LNCaP tumor-bearing mice. The control groups were NPs without aptamers (NP-Apt 0%) (red), and NPs with scrambled nonfunctional Apt (NP-MutApt 10%) (black). Data represent the mean and the standard error of the mean of five mice per group. Data points for NPs-Apt 1% and 5% were statistically significant compared with NP-Apt 0% and NP-MutApt 10% by t test at 95% confidence interval. One-way ANOVA with Fisher's LSD post hoc comparisons at a 95% confidence interval were used for statistical comparisons between groups.

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Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers - PubMed (original) (raw)