Acidity-triggered surface charge neutralization and aggregation of functionalized nanoparticles for promoted tumor uptake

Abstract

A polymeric nanovehicle featuring a histidine-rich surface was developed for tumor-targeted delivery of doxorubicin. By tumor acidity-triggered surface charge neutralization and aggregation of drug-carrying nanoparticles, the drug accumulation within the tumor was significantly enhanced.

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In the last decade, the development of various nanovehicles employed as delivery systems of anticancer drugs has considerably emerged, primarily taking advantage of the enhanced permeability and retention (EPR) effect of tumor tissues.¹ Nevertheless, the accumulation of therapeutic nanoparticles at tumor sites and their uptake by cancer cells is severely impeded by multiple biological barriers and intratumoral interstitial fluid pressure (IFP). In order to overcome these challenges, great efforts have been devoted to the design of functionalized nanocarriers with enhanced stealth effect during blood circulation as well as prolonged retention in tumor by modulating their size and/or surface charge in response to tumor acidic microenvironment (pH_e 6.2–7.0).^2,3 For instance, the gold nanoparticles deliberately coated with zwitterionic self-assembled monolayers have shown highly dispersed colloidal behavior at pH 7.4, thus effectively resisting the uptake by reticuloendothelial system. At pH 6.6, the particles rapidly self-associated into large aggregates which substantially promoted their retention in tumor by enduring tumor IFP.^2b On the other hand, morpholino-decorated polymeric micelles exhibited a promoted uptake by HeLa cells at pH 6.5 compared to that at pH 7.4 by the increased electrostatic attraction with cancer cells via the protonation of the morpholino groups on micelle surfaces at an acidic pH.^3a

In this work, through the hydrophobic anchoring of the tailored-made N-acetyl histidine-modified D-α-tocopheryl polyethylene glycol 1000 succinate (NAcHis–TPGS) on surfaces of poly(lactic-co-glycolic acid) (PLGA)-constituted solid cores laden with DOX in base form, the pH_e-responsive therapeutic polymeric nanoparticles elaborated with histidine-rich surfaces were attained for improved tumor-targeted DOX delivery. Owing to the surface charge neutralization via the pH_e-trigged protonation of histidine residues, which further resulted in colloidal aggregation, the DOX-loaded nanoparticles became more resistant to tumoral IFP and capable of accumulating in tumor site, thus enhancing the uptake by cancer cells (Scheme 1).

Scheme 1 Illustration of acid-triggered surface charge neutralization and aggregation of DOX-loaded NHTPNs for enhanced tumor uptake.

NAcHis–TPGS was attained by the conjugation of amphiphilic TPGS with N-acetyl histidine via dicyclohexylcarbodiimide/4-(dimethyl amino)pyridine mediated esterification.⁴ The detailed synthetic route is illustrated in Fig. S1.† The structure of NAcHis–TPGS was confirmed by ¹H-NMR. The conjugation efficiency was estimated to be greater than 95%.⁴ The DOX-loaded NAcHis–TPGS/PLGA nanoparticles (denoted as DOX-loaded NHTPNs) were prepared by the co-association of PLGA and hydrophobic DOX molecules in aqueous solution containing NAcHis–TPGS at pH 7.4, using a simple nanoprecipitation approach. For comparison, DOX-loaded TPGS/PLGA nanoparticles (DOX-loaded TPNs) and drug-free NHTPNs were also prepared in a similar manner. The pristine NHTPNs, DOX-loaded NHTPNs and TPNs exhibited comparable hydrodynamic diameters and zeta potentials at pH 7.4 (Fig. 1a and b). The high negative values of particle zeta potential at pH 7.4 (Fig. 1b) were observed, mainly as a result of the presence of carboxylic acid groups at one terminal of PLGA chain segments. The TEM image of DOX-loaded NHTPNs at pH 7.4 (Fig. 1c) reveals their morphology in well-dispersed spherical shape. For both NHTPNs and TPNs, high DOX loading efficiencies (ca. 77%) and contents (ca. 7.8 wt%) were attained (Table 1). Superior colloidal stability of the DOX-carrying NHTPNs and TPNs in PBS over a time period of 7 days was also observed (Fig. S2†), demonstrating that TPGS after being conjugated with N-acetyl histidine still retains the excellent performance in stabilizing the PLGA cores at pH 7.4 virtually by its hydrophilic PEG chain segments.

Fig. 1 (a) DLS particle size distribution profiles and (b) zeta potential values of various nanoparticles in aqueous solutions at different pH. TEM images of DOX-loaded NHTPNs at pH 7.4 (c) and 6.5 (d) (scale bars are 200 nm).

Table 1 Dynamic light scattering (DLS) data, drug loading efficiency and capacity of the PLGA-based nanoparticles

Sample	Dh (nm)	PDI	DLE (%)	DLC (wt%)
NHTPNs	50.9 ± 1.3	0.138	—	—
DOX-loaded NHTPNs	45.1 ± 2.5	0.144	77.9 ± 6.0	7.9 ± 0.6
DOX-loaded TPNs	40.9 ± 0.1	0.130	76.3 ± 5.4	7.8 ± 0.9

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Notably, when the medium pH was adjusted from pH 7.4 to 5.0, the zeta potential of NHTPNs laden with DOX was abruptly changed from −15 to +10 mV (Fig. 1b) due to the increased protonation of imidazole groups from histidine residues under weak acidic conditions. Interestingly, when pH was adjusted to 6.5 rather equivalent to pH_e, the DOX-loaded NHTPNs with a zeta potential of ca. −5 mV became easily aggregated, as reflected by their enlarged particle size and size distribution (Fig. 1a and d). This is because the near neutral surfaces of DOX-carrying NHTPNs lack sufficient interparticulate electrostatic repulsion in stabilizing individual nanoparticles. A similar acidity-triggered surface charge conversion and particle aggregation from drug-free NHTPNs was also observed. By contrast, the particle size and zeta potential of DOX-loaded TPNs remained virtually invariant in response to pH changes owing to the void of the sensitivity of the parent TPGS compound to medium pH.

It is postulated that the nanoparticles with surface elaboration by NAcHis–TPGS can remarkably promote their retention and cellular uptake at tumor sites due to the characteristic tumor acidity that triggers surface charge neutralization and thus particle aggregation. Both DOX-loaded NHTPNs and TPNs showed pH-evolved drug liberation behavior as shown in Fig. S3,† obviously due to the increased water-solubility of DOX⁵ and the somewhat accelerated degradation of PLGA cores at pH 6.0. Such an acid-induced drug release of NHTPNs and TPNs could not only prevent drug leakage during their blood circulation to reduce adverse effect on the normal tissues but also achieve the intracellular drug delivery upon endocytosis pathway.

For insight into the effects of pH_e-responsive NAcHis–TPGS surface modification on the cellular uptake of PLGA-based nanoparticles, HeLa cells were incubated with free DOX, DOX-loaded NHTPNs and TPNs, respectively, in different pH environments for 2 h, and then the intracellular DOX concentration was determined. With the cell culture pH being adjusted from 7.4 to 6.0, the intracellular drug concentration of HeLa cells treated with DOX-loaded NHTPNs was largely increased by 8 folds, whereas the intracellular drug level of HeLa cells receiving either free DOX or DOX-loaded TPNs was not remarkably enhanced (Fig. 2a). Moreover, HeLa cells treated with DOX-loaded NHTPNs at pH 6.5 showed a significantly higher intracellular DOX fluorescence intensity in comparison with cells incubated with the same particles at pH 7.4 or DOX-loaded TPNs at pH 7.4 and 6.5 (Fig. 2b). More importantly, the enhanced uptake of DOX-carrying NHTPNs by HeLa cells under weak acidic conditions (pH 6.5 and 6.0) considerably promoted their cytotoxicity (Fig. 2c). In contrast, the cytotoxicity of DOX-loaded TPNs in response to pH reduction was only slightly increased (Fig. 2c). These findings further support our hypothesis in part that the DOX-carrying NHTPNs undergoing the acidity-triggered surface charge transformation could promote not only cellular uptake but also anticancer efficacy. Because of the significant association of hydrophobic DOX molecules with cell membranes rather than nuclei via the passive diffusion,⁶ the cytotoxicity of free DOX against HeLa cells at pH 7.4 was significantly lower than that of DOX at pH 6.5 and 6.0 where the water solubility of DOX was increased to some extent (Fig. 2c).

Fig. 2 (a) Normalized intracellular DOX levels of HeLa cells incubated with free DOX, DOX-loaded NHTPNs and TPNs under different pH conditions for 2 h. (b) Fluorescence images and (c) enhanced cytotoxicity index of DOX-containing formulations against HeLa cells at different pH.

In order to examine the in vivo tumor targeting and biodistribution of DOX-loaded NHTPNs by near-infrared (NIR) fluorescence imaging, the nanoparticles were labelled with the hydrophobic NIR fluorescence dye, IR780. As shown in the in vivo fluorescence images of HeLa tumor-bearing nude mice treated intravenously with different formulations at an identical IR780 dosage at 3 h and 8 h post-injection (Fig. 3a), the NIR fluorescence intensity of tumor treated with the cargo-loaded NHTPNs is significantly higher than that of tumor receiving the TPNs. Also, a stronger IR780 fluorescence signal of the isolated tumor treated with the NHTPNs compared to the tumor receiving the TPNs was observed at 48 h post-injection (Fig. 3b). Notably, based on the findings of immunohistochemical examination of ex vivo tumor sections, in comparison with DOX-loaded TPNs, the DOX-loaded NHTPNs displayed the appreciably higher intratumoral deposition as shown in Fig. 3c. These results strongly confirm that the NHTPNs extravasating from tumor vessels can rapidly and efficiently accumulate at tumor site by their acid-activated surface charge conversion combined with particle aggregation, thus promoting tumor uptake of therapeutic payloads. In order to explore the effects of the acid-triggered surface charge neutralization and aggregation of DOX-loaded NHTPNs on in vivo antitumor efficacy, our next work is to observe the tumor growth of nude mice bearing various styles of tumors treated with drug-loaded NHTPNs and to monitor the survival rate of the treated mice.

Fig. 3 (a) In vivo NIR fluorescence images of HeLa tumor-bearing nude mice receiving i.v. injection of various formulations. (b) Ex vivo NIR fluorescence images of the isolated major organs and tumors 48 h post-injection with different formulations. (c) Fluorescence images of tumor sections from the mice bearing HeLa tumor 48 h post-injection of drug-loaded NHTPNs and TPNs. Tumor cell nuclei and angiogenic blood vessels were stained with DAPI and CD31 markers, respectively.

In summary, the pH_e-responsive functionalized NHTPNs were developed by the surface modification of PLGA nanoparticles with NAcHis–TPGS and the effective delivery of therapeutic agent to tumor sites via NHTPNs by virtue of the tumor acidity-triggered surface charge neutralization and particle aggregation was demonstrated. The results strongly suggest that the NHTPNs developed in this work have great potential of being employed as an advanced nanovehicle for improved tumor-targeted drug transport.

Acknowledgements

Financial supports from Ministry of Science and Technology, Taiwan (MOST 104-2627-M-007-009, 102-2221-E-007-032-MY3 and 103-2221-E-007-023), National Tsing Hua University, Taiwan (105N522CE1) and Hsinchu Mackay Memorial Hospital, Taiwan (MMH-TH-10403) are acknowledged.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra05807h

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