Nanocarriers with dual pH-sensitivity for enhanced tumor cell uptake and rapid intracellular drug release

Abstract

Block polymers are synthesized to prepare nanocarriers with dual pH-sensitivity, which are expected to prolong blood circulation time, reduce systemic toxicity, enhance tumor cell uptake and accelerate intracellular drug release for efficient anti-cancer therapy.

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It is desirable to design nanocarriers which can selectively target diseased sites because in vivo defence mechanisms such as reticuloendothelial system (RES) and protein adsorption in blood, as well as the uptake by normal tissues and cells, can decrease the drug efficiency. Meanwhile, rapid intracellular drug release is particularly important to induce tumor death through sufficient therapeutic drug concentration and overcome the multi-drug resistance (MDR) of multiple treatment cycles in tumor therapy.¹ Recently, the persistent development of stimuli-responsive polymeric carriers for intracellular drug/gene delivery makes them promising in clinical disease therapies.² Thus, it is reasonable to construct nanocarriers with multiple stimuli-sensitivity to simultaneously promote the selective targeting and rapid intracellular drug release.

Compared with the exogenous stimuli, the endogenous stimuli are much better and feasible to be engineered to design drug carriers for optimum in vivo drug therapeutic efficiency. These endogenous stimuli include enzyme degradation, glucose concentration, redox reaction, temperature and pH differences.³ It is noted that pH is one of the most exploited stimuli for drug delivery applications due to the existence of two types of ubiquitous pH differences: slightly acidic pathological tissues (e.g. tumor and rheumatoid arthristis, pH ∼ 6.5) and intracellular acidic compartments (endo-/lysosomes, pH ∼ 5.0) versus normal tissue and blood stream (pH ∼ 7.4). Recently, pH-sensitive charge-conversion strategy has been widely studied to improve the drug delivery efficiency. The highlight is that the conjugated charge-conversional groups are acid-liable and can exhibit reversal charge from negative to positive in response to pH variations. For example, Kataoka et al. fabricated a series of pH sensitive charge-conversional carriers for rapid intracellular protein/gene delivery.⁴ Shen and co-workers found that pH-responsive charge-reversal nanocarriers had improved cytomembrane penetrating ability for targeted anti-cancerous drug/gene delivery.⁵ Wang et al. developed charge-conversional systems to significantly reduce protein adsorption in blood circulation, but efficiently enhance cell adhesion and the followed internalization in response to the slight acidity of tumor tissue.⁶ Chen and co-workers also fabricated drug carriers with pH-sensitive charge transition property based on polypeptides to further improve their biocompatibility.⁷ In all the above systems, nanocarriers with negatively charged surface can significantly prolong the blood circulation time due to the decrease of undesirable clearance by RES. At the specific targeted sites, the nanocarriers will exhibit acidity-induced positive charge, thus they can strongly adhere on negatively charged cell surface through electric interaction and subsequently undergo rapid cell internalization.^5a,5b,7d,8

In view of the demands of anti-cancer therapy, in the above pH-sensitive charge-conversion strategy, it would be ideal to endow nanocarriers with additional rapid intracellular drug release profiles in response to the intracellular acidic compartments, which can further improve drug efficiency by optimizing the release process. Herein, to demonstrate this concept, poly(2-diisopropylaminoethyl methacrylate)-block-poly(2-aminoethyl methacrylate hydrochloride) (PDPA-b-PAMA) was synthesized according to our previous report^1d and the amino groups in PAMA block were further functionalized using 2,3-dimethykmaleic anhydride (DMMA) (Fig. S1, ESI†). The PDPA-b-PAMA backbone (MW 9800, PDI 1.37, i.e., having 24 DPA repeated units and 21 AMA repeated units) is capable of rapid intracellular drug release in response to the acidity of endo-/lysosomes.^1d The DMMA functionalized PDPA-b-PAMA (PDPA-b-PAMA/DMMA) can self-assemble into nanomicelles with dual pH-sensitivity. Once the doxorubicin (DOX)-loaded micelles are accumulated in the tumor tissue by enhanced permeability and retention (EPR) effect, the first pH-sensitivity happens that DMMA is gradually hydrolyzed from the carriers, which will exhibit positively charged surface in response to the slight pH change to facilitate their adhesion on cell surface through electric interaction and the followed endocytosis. Subsequently, after carriers being trapped into intracellular acidic compartments, the second pH-sensitivity works that the disassembled block copolymers rapidly release DOX to induced tumor cell death (Scheme 1).

Scheme 1 Illustration of DOX-loaded micelles with dual pH-sensitivity for anti-cancer therapy.

The successful functional modification of PDPA-b-PAMA/DMMA was confirmed by ¹H NMR analysis (Fig. S2 and Experimental section, ESI†). In pH 7.4 aqueous solution, PDPA-b-PAMA/DMMA can self-assemble into nanomicelles with hydrophobic PDPA aggregated inner core and hydrophilic PAMA/DMMA outer corona (Fig. S3, ESI†). The thermodynamic stability of nanomicelles was investigated through measuring its critical micelle concentration (CMC) by pyrene fluorescence technique at pH 7.4. The CMC value of polymeric self-assemblies is 4.4 × 10⁻³ mg mL⁻¹ (Fig. S4, ESI†). The low CMC value indicates sufficient stability to reduce drug loss during the blood circulation, which is potential for in vivo drug delivery under high dilution.

In order to study the dual pH-sensitivity, DLS was firstly employed to detect the hydrodynamic diameter (D_h), size distribution and zeta potential at different pH values. Different from PDPA-b-PAMA self-assembly, which is the control sample exhibiting positive zeta potential (+13.5 to +19.1 mV) at all the pH values ranging from 8.0 to 5.5, the zeta potential of PDPA-b-PAMA/DMMA continuously increase from negative to positive along with the decrease of pH value (Fig. 1A). Notably, it shows negative surface charge (−9.9 mV) at pH 7.4, but positive surface charge (+12.25 mV) at pH 6.5. This pH-induced charge reversal phenomenon of the surface charge of PDPA-b-PAMA/DMMA, i.e., from negative to positive, confirms the first pH-sensitivity that DMMA conjugates hydrolyze from the block copolymer at slight acidic environment.

Fig. 1 Zeta potential (A) and average D_h size (B) studies of PDPA-b-PAMA and PDPA-b-PAMA/DMMA self-assemblies at different pH (0.5 mg mL⁻¹; 37 °C). Data are presented as the average ± standard deviation (n = 3). TEM micrographs of PDPA-b-PAMA/DMMA at pH 7.4 (C), 6.5 (D) and 5.0 (E). The scale bar in TEM micrographs is 50 nm. BSA adsorption on the polymeric self-assemblies at different pH (7.4, 6.5 and 5.0). Data are presented as the average ± standard deviation (n = 6).

In the following D_h measurements, both PDPA-b-PAMA and PDPA-b-PAMA/DMMA self-assembles exhibit mean D_h size (PDPA-b-PAMA/DMMA: 145 ± 3.6 nm at pH 7.4 and 154 ± 2.5 nm at pH 6.5) and relatively narrow size distribution at pH ≥ 6.5 (Fig. 1B and Fig. S5, ESI†). However, both samples do not exhibit mean D_h size at pH ≤ 6.0. This pH-induced disassembly is attributed to the second pH-sensitivity which makes the amphiphilic block copolymer transfer to double hydrophilicity due to the significant protonation of PDPA block at pH ≤ pK_a (∼6.3). This pH-sensitive disassembly of micelle can lead to rapid cargo release in response to intracellular acidic compartments, which is quite essential to kill pathological cells and overcome multi-drug resistance in tumor therapy.

In order to further confirm the pH-sensitivity of PDPA-b-PAMA/DMMA self-assemblies, TEM was used to visually observe the morphologies at typical pH conditions (pH 7.4, 6.5 and 5.0). PDPA-b-PAMA/DMMA can self-assemble into sphere micelles with an average dehydrated diameter around 30 nm and 35 nm at pH 7.4 and 6.5, respectively (Fig. 1C and D). The slight size increase of micelles at relatively low pH may be caused by the expansion of partially protonated PDPA block.^1c In contrary, no obvious self-assembly is observed at pH 5.0 (Fig. 1E and S3, ESI†) due to the lack of self-assembling driving force, i.e., the fully protonated PDPA block leads to a hydrophilicity variation of block copolymer from amphiphilic to double hydrophilic. Thus, the visual observation of micellar disassembly responding to the simulative intracellular acidity of endo-/lysosome confirms the second pH-sensitivity of PDPA-b-PAMA/DMMA self-assembly.

To inspect the first pH-sensitivity for potential in vivo application, the protein adsorption of micelles were evaluated using bovine serum albumin (BSA) as a model protein at different pH (7.4, 6.5 and 5.0) (Fig. 1F). PDPA-b-PAMA sample strongly interacts with BSA protein at all measured pH values, even up to 96.4% at pH 5.0. In contrast, PDPA-b-PAMA/DMMA sample exhibits minimal BSA adsorption (8.2%) at pH 7.4 due to its negatively charged surface, but large amount of BSA (more than 71.8%) is adsorbed onto its positively charged surface through strong electric interaction at pH ≤ 6.5. This phenomenon is well in agreement with the pH induced charge conversion detected by DLS. At pH 7.4, PDPA-b-PAMA/DMMA sample remains negative surface charge and exhibits protein-resistant property for long blood circulation. On the other hand, with the decrease of pH value, it gradually transfers to positive surface charge and shows enhanced interaction with protein, which could improve its cell adhesion and internalization.^6e,9

The hydrophobic desalted DOX was used to investigate the drug encapsulation and drug release behaviors of PDPA-b-PAMA/DMMA micelle. DOX is loaded into the hydrophobic core of polymeric micelles through physical encapsulation. The DOX-loaded carrier shows relatively high drug encapsulation ability (loading efficiency (LE): 15.7%; encapsulation efficiency (EE): 81.1%, Table S1, ESI†) and uniform size distribution (187.6 ± 12.4 nm detected by DLS, TEM, Fig. S6, ESI†). Then, in vitro drug release profiles are investigated in different pH phosphate buffer solutions (PBS, 0.01 M, pH 7.4, 6.5 and 5.0) (Fig. 2A). Because of its particularly sturdy hydrophobic core structure, the cumulative release of DOX from carrier is less than 15% and 25% within 36 h at pH 7.4 and 6.5, respectively. However, when the pH value decreases to 5.0, more than 87% of loaded drug is released from carrier within 36 h, which indicates its sufficient sensitivity to the intracellular acidic compartments. Thus, the pH-dependent DOX release property can reduce premature drug loss during the blood circulation and enhance intracellular drug release in acidic compartments, which could not only reduce the side effects for normal cell, but also promote the therapeutic efficiency for tumor cell kill.

Fig. 2 Release profiles of DOX from PDPA-b-PAMA/DMMA micelles at different pH (A). Confocal images of HeLa cells incubated with DOX-loaded PDPA-b-PAMA/DMMA micelles with a concentration of 0.5 mg mL⁻¹ for 4 h. The scale bar is 100 μm. Blue: Hoechst 33342 stained nuclei (B); green: Lysotracker Green DND-26 stained acidic compartments (C); red: DOX (D); merged fluorescence (E).

Consequently, the intracellular drug distribution was visually monitored by observing HeLa cells cultured with DOX-loaded PDPA-b-PAMA/DMMA by CLSM. In order to better simulate the tumor cell endocytosis at the slight acidic pH environment of extracellular matrix of tumor tissue, the pH value of culture media is artificially adjusted to 6.5. After only 4 h incubation with HeLa cells, strong DOX red fluorescence is clearly observed in the DOX fluorescence channel scanning (Fig. 2D). Moreover, the DOX red fluoresce completely overlaps with the blue fluoresce of Hoechst 33342 stained nuclei in the merged status (Fig. 2E), which reveals that DOX is burst release in response to the low pH of acidic compartments and subsequently accumulate into the nuclei of tumor cells. In addition, the fast intracellular drug release behavior from PDPA-b-PAMA/DMMA is consistent with the in vitro drug release studies.

To verify the feasibility of PDPA-b-PAMA/DMMA carrier, in vitro cytotoxicity was evaluated by standard MTT assay. HeLa cells are treated with a series concentration of blank polymeric micelles for 24 h. The results demonstrate that the polymeric carrier has very low cytotoxicity (higher than 80% of cell viability) even at fairly high concentration up to 100 μg mL⁻¹ (Fig. 3). Thus, it suggests that PDPA-b-PAMA/DMMA carrier is biocompatible for drug delivery.

Fig. 3 Cell viability of PDPA-b-PAMA/DMMA micelles at different concentrations (pH 7.4). HeLa cells are incubated at 37 °C for 24 h. Data are presented as the average ± standard deviation (n = 6).

In summary, the PDPA-b-PAMA/DMMA self-assembly with dual pH-sensitivity integrates surface charge-conversion and rapid intracellular drug release properties, which are expected to have superior performances including long blood circulation, low cytotoxicity, tumor targeting, strong cell adhesion, rapid cell endocytosis and fast intracellular drug release for efficient tumor kill. Thus, it can be a potential candidate as efficient drug carrier for tumor therapy.

Acknowledgements

Financial support from the National Natural Science Foundation of China (51322303) and Foundations of Sichuan Province (2012JQ0009) are gratefully acknowledged.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Including synthesis, characterization and other experimental details. See DOI: 10.1039/c4ra05270f

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