A facile way to fabricate pH-sensitive charge-conversion polymeric nanoparticles with tunable pH conversion point

Abstract

pH-Sensitive charge-conversion polymeric nanoparticles could significantly enhance drug bioavailability due to improved tumor cell internalization. However, the extracellular pH of tumors is significantly different for tumors of different histology and volume. To address this point, a series of pH-sensitive charge-conversion succinyl chitosan–O–poly(ε-caprolactone) (SC–O–PCL) nanoparticles with a tunable pH conversion point were fabricated through a facile method. Surface zeta potential of SC–O–PCL nanoparticles can change from negative (physiological pH) to positive in acidic conditions. The positive charges on the nanoparticles were beneficial to tumor cell internalization, leading to enhanced drug bioavailability. Also, the pH conversion point can be modulated in a wide range to match the varied tumor extracellular pH. Furthermore, the nanoparticles can encapsulate the antitumor drug doxorubicin (Dox) effectively and release Dox quickly in response to an acidic environment. This kind of smart nanoparticles may be a feasible candidate for individual tumor therapy.

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Introduction

Chemotherapy is an important method for clinical treatment of cancers. However, random distribution of antitumor drugs in the human body leads to low drug bioavailability and serious side-effects. Polymeric nanoparticles, used as an antitumor drug carrier, could accumulate into tumor tissue through an enhanced permeation and retention (EPR) effect.¹ The incorporation of antitumor drug into polymeric nanoparticles has already shown improved drug bioavailability, reduced drug resistance and enhanced therapeutic efficacy.^2,3 It is well-known that positively charged nanoparticles can reliably stick to cell membranes due to attractive electrostatic force, which significantly increases the cellular internalization efficiency and promotes the drug accumulation in tumor cells.^4,5 For example, Xia et al.⁵ investigated the cellular uptake of gold nanoparticles with different types of surface charge and results showed that positively charged gold nanoparticles exhibited much higher cellular internalization efficiency than those with neutral or negatively charged surface. However, positive charges on the nanoparticle surface could also facilitate the interaction between the nanoparticles and non-specific proteins during the circulation period, resulting in rapid elimination by the reticuloendothelial system (RES).⁶ On the other hand, nanoparticle with neutral or negatively charged surface could resist non-specific protein adsorption and avoid rapid elimination by the RES, leading to long circulation time.⁷ Charge-conversion nanoparticle could be used to shield positive charge during drug delivery.

Significant differences between the microenvironment of tumor and adjacent normal tissue could be explored as trigger to design charge-conversion nanoparticle for tumor therapy. For example, the pH of normal tissue is about 7.4, while the tumor tissue is acidic with pH = 6.0–7.0. During the last decade, numerous pH-sensitive charge-conversion nanoparticles utilizing variations in pH values had been developed.^8–10 For example, pH cleavage amide bond was introduced into polymer and the breakage of amide bond in acidic tumor microenvironment switched the surface charge of nanoparticle from negative to positive, leading to enhanced cellular uptake.⁸ However, it should be noted that the tumor extracellular pH is significantly different for tumors of different histology and volume.^11,12 Hence, it is essential for drug carriers having controllable pH conversion point for individual tumor therapy.

In this work we reported a facile way to fabricate a series of pH-sensitive charge-conversion nanoparticles with tunable pH conversion point based on synthesized zwitterionic chitosan derivatives. The derivatives consisted of chitosan, poly(ε-caprolactone) (PCL) and succinic anhydride. The formed nanoparticles were negatively charged at physiological pH. Surface zeta potential of nanoparticles switched from negative to positive quickly in acidic condition. And more importantly, the pH conversion point could be adjusted simply by modulating the degree of substitution of succinic anhydride in the polymer. This type of smart nanoparticles may have potential application as antitumor drug carrier for individual tumor therapy.

Experimental

Materials

Chitosan (CS, MW 5000, degree of deacetylation was 90%) was purchased from Yuhuan Ocean Biomaterial Co. Ltd. PCL (M_n ∼ 4500) was synthesized in our laboratory using benzyl alcohol as an initiator and Sn(OCt)₂ as a catalyst. Trifluoroethanol (TFE) and succinic anhydride (SA) was supplied by J&K. Rhodamine B isothiocyanate (RBITC) was purchased from Sigma. Doxorubicin (Dox) hydrochloride (>99%) was obtained from Hangzhou Haida Pharmaceutical Chemical Co. Ltd. All other chemicals and solvents were of analytical grade and used as received. Human mammary cancer line Bcap 37 cells, purchased from ATCC, were maintained in RPMI 1640 medium and supplemented with 10% fetal bovine serum, 100 μg ml⁻¹ streptomycin and 1% antibiotic/antimycotic solution.

Synthesis of SC–O–PCL

SC–O–PCL was synthesized through two-step route. Firstly, CS–O–PCL was synthesized according to our previous report using sodium dodecyl sulphate (SDS)–chitosan complex (SCC) as intermediate.¹³ Then, CS–O–PCL was further reacted with succinic anhydride to obtain SC–O–PCL. Briefly, 0.1 g CS–O–PCL was dispersed in 5 ml TFE, and a certain amount of succinic anhydride dissolved in acetone was added. The reaction was continued for 24 h at 40 °C. Then the reaction product was precipitated into methanol. The precipitates were collected by centrifugation, washed by methanol, and finally vacuum-dried to yield SC–O–PCL.

Preparation of SC–O–PCL nanoparticle

The aqueous nanoparticle solution was prepared by traditional dialysis method. 25 mg SC–O–PCL was dissolved in 8 ml TFE/acetic acid/H₂O (v/v/v = 5 : 1 : 2) solution. The solution was dialyzed against pH = 7.4 phosphate buffer solution (PBS) with a cellulose membrane (MWCO 3500) at room temperature for 2 days to yield a transparent solution.

Determination of critical aggregation concentration (CAC)

Pyrene fluorescence method was used for CAC determination of SC–O–PCL nanoparticle.¹⁴ Fluorescence spectra were recorded on a fluorescence spectrophotometer (F-4600, Hitachi). Samples for fluorescence measurement were prepared by adding certain amount of pyrene into the aqueous nanoparticle solution. And the pyrene concentration in the solution was 6.0 × 10⁻⁷ M. For the measurement, the slit widths for both excitation and emission sides were fixed at 2.5 nm and the emission wavelength was set at 372 nm. The dependence of I₃₃₉/I₃₃₅ on the concentration of nanoparticles solution was plotted. The CAC was taken as the intersection of the tangent to the curve at the inflection with the horizontal tangent through the points at low solution concentration.

Stability evaluation of SC–O–PCL nanoparticle

Stability of SC–O–PCL nanoparticle was evaluated by measuring the changes of diameter of the system with incubation time.

Furthermore, pyrene was used as a fluorescent probe to monitor the integrity of SC–O–PCL nanoparticle. The concentration of SC–O–PCL nanoparticle solution was set at 1 mg ml⁻¹ and the pyrene concentration was 6.0 × 10⁻⁷ M. The emission wavelength was set at 372 nm and the excitation spectra were recorded. The polarity of the PCL core was characterized by the intensity ratio of I₃₃₉/I₃₃₅.^15,16

Preparation of Dox-loaded SC–O–PCL nanoparticle

Dox-loaded nanoparticle was fabricated via an emulsion method.¹⁷ 5 mg Dox hydrochloride was dissolved in 1 ml CHCl₃ containing 10 μl triethylamine. The solution was stirred overnight to remove HCl, and then 200 μl the above solution was added dropwise into 5 ml SC–O–PCL solution (1 mg ml⁻¹). The mixed solution was stirred in darkness overnight and the CHCl₃ was removed by evaporation, and unloaded Dox was removed by filtration through 0.45 μm filters to yield Dox-loaded nanoparticles.

Lyophilized SC–O–PCL nanoparticle (2 mg) were dissolved in 5 ml TFE/acetic acid/H₂O (v/v/v = 5 : 1 : 2) solution, and the Dox concentration was determined using a UV-vis spectroscopy (Lambda 35, Perkin Elmer) at 483 nm. The loading content (LC) and entrapment efficiency (EE) of Dox were calculated according to the following equations:

Loading content (LC) = (W₁/W₂) × 100% (1)

Entrapment efficiency (EE) = (W₁/W₃) × 100% (2)

here W₁ and W₃ were the weight of the loaded Dox within the nanoparticles and the initially added Dox, and W₂ was the weight of SC–O–PCL nanoparticles.

In vitro release of Dox

4 ml Dox-loaded nanoparticle solution was dialyzed with a cellulose tube (MWCO 3500) against 50 ml phosphate buffer medium. At scheduled time, 4 ml of release medium was withdrawn and replaced with an equal volume of fresh medium at 37 °C. The amount of released Dox was determined with UV-vis spectroscopy at 483 nm.

Cytotoxicity test

In vitro cytotoxicity of SC–O–PCL nanoparticles was evaluated by MTT assay. Bcap 37 cells were seeded in a 96-well plate with a density of 1 × 10⁴ cells per well and incubated in a culture medium (RPMI 1640, 10% FCS) for 24 h at 37 °C and 5% CO₂. Then 22 μl serials of polymer solution was added per well. The cells were then incubated for 24 h at 37 °C. Afterward, the medium was replaced by 200 μl fresh 1640 culture medium and the cells were incubated for another 24 h. Then 22 μl MTT solutions (5 mg ml⁻¹) were added per well. After another 4 h incubation, medium was replaced by 200 μl dimethylsulfoxide per well. Measurement was performed using a microplate reader at a wavelength of 570 nm. 1640 medium and polyethylenimine (25 kDa, 10 mg ml⁻¹) were used as negative and positive controls, respectively.

The in vitro cytotoxicity of the Dox-loaded nanoparticles was evaluated following the above described procedures.

Labeling of SC–O–PCL nanoparticle with RBITC

RBITC (10 mg) was dissolved in the admixture of pH 9.0 carbonate buffer (3 ml) and deionized water (7 ml). Then the solution was introduced into the aqueous SC–O–PCL nanoparticle solution. The mixed solution was incubated in darkness at room temperature overnight, and then dialyzed against pH 7.4 PBS for 5 day and deionized water until no RBITC was detected in the dialysis solution. The final product was obtained by freeze-drying and stored in refrigerator.

Cell internalization of SC–O–PCL nanoparticles

Bcap 37 cells were cultured on a 6-well plate containing a glass slide in each well (the slide was pre-laid on the bottom) and incubated overnight. Then the cell culture medium was replaced with the fresh medium containing SC–O–PCL-B nanoparticles with or without pretreatment in pH 6.0 buffer for 12 h. The nanoparticles without Dox-loaded contained equivalent amount of RBITC-labeled SC–O–PCL (10 μg ml⁻¹). The cells were incubated at 37 °C and 5% CO₂ for 2 h, washed thoroughly with PBS 3 times, fixed with 4% formaldehyde and then observed with confocal laser scanning microscopy (CLSM, Nikon C2).

Characterization methods

¹H NMR spectra were obtained by a Bruker DMX-500 NMR spectrometer operating at 500 MHz. The dynamic diameter (D_h) and zeta potential of the nanoparticle were recorded using a Malvern nanoZS90 analyzer. All nanoparticle solutions had a final concentration at 1 mg ml⁻¹. And prior to the light scattering test, all the samples were filtered through 0.45 μm filters. Transmission electron microscopy (TEM) images were obtained by a JEOL JEM 1230 electron microscope operating at an acceleration voltage of 80 kV. Samples were deposited from 1 mg ml⁻¹ nanoparticle solution onto copper grids coated with carbon. Water was allowed to evaporate at atmospheric pressure and room temperature. And 1% uranyl acetate solution was used to stain the grid.

Results and discussion

Synthesis and characterization of SC–O–PCL copolymer

The synthetic route of SC–O–PCL was shown in Fig. 1. Low molecular weight chitosan was used in this work due to its good water solubility. Using sodium dodecyl sulfate–chitosan complex (SCC) as intermediate, PCL was region-selectively grafted onto C6 site of the polysaccharides group in a mild and homogenous reaction condition. In our previous study,¹³ it was confirmed that amino groups did not participate the conjugation reaction and SDS could be completely removed from the final products by precipitating into tris(hydroxymethyl)aminomethane (Tris) solution.

Fig. 1 Synthetic route of SC–O–PCL and schematic illustration of charge-conversion nanoparticles with tunable pH conversion point.

The structures of intermediates and final product were characterized by ¹H NMR, as shown in Fig. 2. Peaks assigned to each segment were clearly marked. The presence of methylene peaks at 3.81, 1.95, 1.25, 1.05 ppm confirmed the successful grafting of PCL onto CS and the disappearance of all the SDS peaks at 0.85, 1.25, 1.5, 3.65 ppm showed that SDS was completely removed. PCL grafting level was calculated by integral ratio of the peak a to the peak 2 and it was about 9.8%. Then succinic anhydride (SA) was reacted with amino group of CS–O–PCL to yield SC–O–PCL. Degree of substitution of succinic anhydride (DS_SA) was calculated by integral ratio of the peak h, h′ to the peak 2 and it could be modulated by changing the feed ratio of SA/CS–O–PCL. With the increase of feed ratio, there was an increase of DS_SA (Table 1). For example, when the feed ratio increased from 0.025 to 0.125, the DS_SA increased from 5.3% to 15.1%.

Fig. 2 ¹H NMR spectra of SCC in DMSO-d6, CS–O–PCL and SC–O–PCL-B in trifluoroacetic acid-d1/D₂O (v/v = 1 : 1).

Table 1 Characteristics of SC–O–PCL copolymers

Samples	Feed ratio^a	DSSA (%)
a Weight ratio between SA and CS–O–PCL.
SC–O–PCL-A	0.025	5.3
SC–O–PCL-B	0.075	9.4
SC–O–PCL-C	0.125	15.1

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Characterizations of SC–O–PCL nanoparticles

The self-assembly behavior of the copolymers was studied by fluorometry, dynamic light scattering (DLS) and transmission electron microscope (TEM). The critical aggregation concentrations (CAC) of nanoparticle solutions were determined from the fluorescence excitation spectra by using pyrene as a fluorescence probe. Pyrene exhibits a peak shift in its excitation spectrum when incorporated into the hydrophobic core of nanoparticle.¹⁸ Fig. 3 plotted the fluorescent intensity ratios of I₃₃₉/I₃₃₅ in excitation spectra and the data were calculated and summarized in Table 2. The CACs of SC–O–PCL were on the similar order of magnitude as those of other chitosan based copolymer reported previously.^19,20 Such low CAC could efficiently reduce the loss of drug during the delivery process under high dilution conditions. And the increase of DS_SA lowered the CAC of the copolymers. Higher DS_SA meant more carboxyl groups in the copolymer, which may affected the water solubility of the chitosan due to electrostatic interaction between carboxyl and amino groups.

Fig. 3 Plots of I₃₃₉/I₃₃₅ vs. log C for SC–O–PCL nanoparticles.

Table 2 Characteristics of SC–O–PCL nanoparticles

Samples	CAC (mg ml^−1)	Dh^a (nm)	Dh^b (nm)	LC (%)	EE (%)
a Diameters of blank SC–O–PCL nanoparticles.b Diameters of Dox-loaded SC–O–PCL nanoparticles.c The polydispersity of the nanoparticles.
SC–O–PCL-A	1.0 × 10^−2	25 (0.234)^c	33 (0.278)^c	11.8	61.1
SC–O–PCL-B	5.1 × 10^−3	26 (0.279)	38 (0.303)	12.6	63.8
SC–O–PCL-C	2.8 × 10^−3	31 (0.218)	42 (0.285)	13.9	66.7

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The average dynamic diameter (D_h) of the nanoparticles in pH 7.4 PBS buffer solution was shown in Fig. 4 and Table 2. The mean diameter of the blank nanoparticles was within a narrow range of 25–35 nm. Increase of DS_SA led to a slightly enlargement in particle size, which may due to the electrostatic repulsive forces of the carboxyl groups. The average diameter of the nanoparticles slightly increased after incorporation of Dox (Table 2). Similar result was also reported before,²¹ which may due to the enhanced hydrophobicity of Dox-loaded PCL core. It was reported that nanoparticles with diameter ∼30 nm showed enhanced tumor penetration and antitumor efficiency in tumor therapy.²² So the obtained drug-loaded nanoparticle was suitable nanocarrier for antitumor drugs. Meanwhile, spherical nanoparticles with a narrow size distribution were formed for all the SC–O–PCL copolymers in the buffer solution. Typical size distribution and TEM image of Dox-loaded SC–O–PCL-B nanoparticles were shown in Fig. 4.

Fig. 4 (a) Size distribution and (b) TEM image of Dox-loaded SC–O–PCL-B nanoparticle.

Then the stability of the nanoparticles in pH 7.4 PBS buffer solution was evaluated by monitoring the variation of diameter with the incubation time. All the nanoparticles maintained their size at the end of the study (Fig. 5), which meant that the SC–O–PCL nanoparticles were stable and no aggregate formed in PBS solution. The good solubility of the low molecular weight chitosan in PBS buffer solution, as well as the electrostatic repulsions, prevented the aggregation of the nanoparticles.

Fig. 5 Change of diameter of SC–O–PCL nanoparticles with incubation time.

Charge-conversion of SC–O–PCL nanoparticles

The charge-conversion ability of the SC–O–PCL nanoparticles was investigated by a zeta potential analyzer. Higher amount of carboxyl groups meant lower zeta potential of nanoparticles in pH 7.4 PBS buffer. The zeta potential was −7.5 mV for SC–O–PCL-C nanoparticle and −1.9 mV for SC–O–PCL-A nanoparticle. All the nanoparticles showed smart charge-conversion properties when pH of medium was changed from basic to acidic. And the charge conversion occurred quickly compared with the amide bond cleavage-based charge-conversion system.²³ Meanwhile, the pH conversion point could be modulated simply by changing the DS_SA of SC–O–PCL, as shown in Fig. 6. By increasing the DS_SA, the pH conversion point was reduced. Since the tumor extracellular pH is significantly different due to different tumor histology and tumor volume,^11,12 the obtained smart nanoparticles with tunable pH conversion point could be utilized for treatment of individual tumor therapy. And the charge-conversion ability of SC–O–PCL nanoparticles indicated that the nanoparticles was negatively charged during the circulation period (pH = 7.4) and would not interact with anionic plasma proteins, resulting in high resistance to non-specific protein adsorption. When the nanoparticles were accumulated in the acidic tumor tissue via EPR effect, charge conversion happened and the positively charged nanoparticles strongly interacted with tumor cell membranes, leading to high efficient cellular internalization.

Fig. 6 Charge-conversion ability of SC–O–PCL nanoparticles.

Furthermore, the pH sensitivity of SC–O–PCL nanoparticles was studied using pyrene fluorometry method (Fig. 7a). Pyrene is a useful indicator for determining the CAC and stability of nanoparticles. The I₃₃₉/I₃₃₅ ratio represented the inner core's hydrophobicity and it decreased when the system was destabilized.^15,16 SC–O–PCL nanoparticles showed excellent stability since the I₃₃₉/I₃₃₅ ratio was quite stable in a wide range of pH from 5.0 to 7.4. The insignificantly changed diameter also confirmed the stability of the nanoparticles (Fig. 7b).

Fig. 7 (a) The intensity ratio I₃₃₉/I₃₃₅ of pyrene excitation spectra in SC–O–PCL nanoparticles solution with medium pH; (b) the change in the D_h of SC–O–PCL nanoparticles with medium pH.

In vitro release of Dox

Dox was used as model drug to evaluate the controlled release ability of SC–O–PCL nanoparticles. Dox was incorporated into nanoparticles using emulsion method. The loading content (LC) and the entrapment efficiency (EE) were summarized in Table 2. Both of them increased with the increase of DS_SA of SC–O–PCL. Higher content of carboxyl group could enhance interactions between Dox and copolymer, which led to higher LC and EE. In vitro release profiles of Dox from the SC–O–PCL nanoparticles were shown in Fig. 8. It was reported that Dox may form an ionic complex with carboxyl group of N,O-carboxymethyl chitosan.²⁴ Hence the increase of DS_SA of copolymer resulted in a slight decrease in the cumulative release amount of Dox. As shown in Fig. 8b, the release of Dox was significantly faster at pH 5.0 than that at pH 7.4. Approximately 85% of Dox was released from SC–O–PCL-B nanoparticle at pH 5.0 in 24 h. In contrast, only 35% of Dox was released at pH 7.4. Similar pH-sensitive release behaviour of Dox was also reported by others.^17,25 Protonation of Dox and the acid-catalyzed cleavage of the azomethine bond in Dox–Dox accelerated the diffusion ability of Dox, resulting in pH-sensitive release profiles.^17,26 Dox was released slowly from the nanoparticles under extracellular condition. But after entering tumor tissue (pH 6.0–7.0) or endosome/lysosome (pH 5.0–6.0),²⁷ Dox was quickly released. This accelerated drug release at lower pH allowed the drug-loaded nanoparticles to achieve the preferential release of drug at tumor tissue.

Fig. 8 In vitro release profiles of DOX (a) from SC–O–PCL nanoparticles incubated in PBS (pH 7.4) at 37 °C and (b) from SC–O–PCL-B at different pH.

Cell internalization and in vitro cytotoxicity

RBITC-labeled SC–O–PCL nanoparticles were prepared through the conjugation reaction between the isothiocyanate group of RBITC and the exposed amino/hydroxyl groups of the SC–O–PCL nanoparticles. Then the cell internalization of nanoparticles was studied by observing Bcap-37 cells co-cultured with RBITC-labeled SC–O–PCL-B nanoparticles under CLSM (Fig. 9). After 2 h incubation, strong red fluorescence was observed in the cells incubated with pretreated RBITC-labeled SC–O–PCL-B nanoparticles. In contrast, much weaker red fluorescence was found in the cells treated with nanoparticles unpretreated in pH 6.0 buffer. Small size (∼30 nm) could facilitate the cell internalization of nanoparticles. The main difference in the intracellular distribution was attributed to the pH-sensitive charge-conversion of SC–O–PCL nanoparticles. Positively charged nanoparticles in acidic condition would enhance the cell internalization efficiency.

Fig. 9 CLSM images of Bcap 37 cells incubated with RBITC-labeled SC–O–PCL-B with or without pretreatment in pH 6.0 buffer solution for 2 h.

In vitro cytotoxicity of the SC–O–PCL nanoparticles was evaluated by MTT assay. In our previous work, CS–O–PCL showed negligible cytotoxicity.¹⁹ Similar results were obtained for SC–O–PCL copolymers. The cell viability of the SC–O–PCL nanoparticles was higher than 80% even at fairly high concentration up to 500 μg ml⁻¹ (Fig. 10a). The cytotoxicity of Dox-loaded nanoparticles was further explored. SC–O–PCL-B nanoparticles were pretreated in pH 6.0 buffer solution for 12 h in order to liberate the incorporated Dox. Compared with original nanoparticles, the pretreated nanoparticles displayed much higher toxicity towards tumor cells, which was close to that of free Dox in the case of high Dox concentration. Most part of the incorporated Dox was released and the surface charge switched from negative to positive after pretreatment of SC–O–PCL-B nanoparticles in pH 6.0 buffer solution, resulting in the elevation of the cytotoxicity.

Fig. 10 In vitro cytotoxicity of (a) SC–O–PCL nanoparticles after 24 h incubation with Bcap-37 cells and (b) Dox-loaded SC–O–PCL-B with different pretreatment (n = 4).

Conclusions

A series of pH-sensitive charge-conversion zwitterionic chitosan derivatives (SC–O–PCL) with different DS_SA were conveniently synthesized by a facile route using SCC as intermediate under mild and homogeneous conditions. Spherical nanoparticles with the average diameter ranged from 25 to 35 nm were formed and the diameters were slightly increased to 30–45 nm after drug-loading. Their surface charge could convert from negative to positive in response to tumor acidity. More importantly, the pH conversion point could be simply regulated by changing the DS_SA of the SC–O–PCL copolymers. Dox was efficiently encapsulated into SC–O–PCL nanoparticles and released quickly in acidic conditions. Suitable size and charge-conversion ability of the nanoparticles could endow them long blood circulation time and enhanced tumor cell internalization. Thus, the obtained smart nanoparticles may have potential use in efficient tumor therapy.

Acknowledgements

The authors thank Peng Liu and Hongliang Jiang for their helpful discussion.

Notes and references

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