Article pubs.acs.org/Macromolecules
Oxidation and Acid Milieu-Disintegratable Nanovectors with Rapid Cell-Penetrating Helical Polymer Chains for Programmed Drug Release and Synergistic Chemo-Photothermal Therapy Yu Chen, Zhi-Huang Zhang, Xin Han, Jun Yin,* and Zong-Quan Wu* Department of Polymer Science and Engineering, School of Chemistry and Chemical Engineering, Hefei University of Technology and Anhui Key Laboratory of Advanced Functional Materials and Devices, Hefei 230009, China S Supporting Information *
ABSTRACT: Polymeric assemblies are distinguished by ease of preparation, high drug-loading content, and long circulation time compared to small molecular, making them quite promising for cancerous diagnosis and therapy. However, the therapeutic efficacy of traditional nanocarriers with random coil surface is always proved to be less effective because of the existence of several systemic and cellular barriers or the low tissue penetration from nanocarrier itself. To fill this gap, we report a new class of oxidation and pH dual-responsive amphiphilic triblock copolymer: poly(L-lactic acid)(-IR780)-b-hydrophobic poly(phenyl isocyanide)-b-hydrophilic poly(phenyl isocyanide) (PLLA(-IR780)-HBPPI-HPPPI). In neutral aqueous solution, the copolymers could form onion-like spherical micelles with diameter of ∼84 nm and consisting of PEGylated single left-handed helical PPI corona, endowing them rapid cell membrane permeability and internalization (10−20 min) that had an analogous effect of cell penetrating peptides (CPPs). Moreover, the phenylboronic pinacol ester contained in the hydrophobic interlayer was stable under neutral and weak acid milieu and thus could minimize the premature drug leakage and systemic cytotoxicity. Upon exposure to H2O2, the interlayer was oxidized rapidly and accompanied by a hydrophobic−hydrophilic transition, which resulted in the releasing of encapsulated drugs and creating interconnected hydrophilic channels to the inner PLLA core at the same time. An enhanced drug release from PLLA core was then achieved by the acid-triggered micelle degradation. The degradation rates of micelles and release rates of drugs could be easily tuned by changing the concentration of H2O2 and the acidity. The hyperthermia induced by the micelles could increase to as high as ∼48 °C upon near-infrared (NIR) light irradiation (808 nm, 1 W cm−2) due to the introduction of NIR absorptive IR780 dyes. Combined with the effect of chemotherapeutics, fatal and irreversible damage to cancer cells was observed. The primary objective of this research was to address the growing need for an effective/rapid drug delivery system and programmed/sustained ondemand drug release. We speculate that the newly developed multifunctional integrated micelles with combined advantages can potentially be utilized as a promising approach to disease diagnosis and therapy.
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cular diseases, and several severe pathological disorder.16−18 In recent years, a series of oxidation-responsive materials have been emerged and showed great potential for H2O2 detection and targeted drug delivery.19−27 Phenylboronic pinacol ester, due to their specificity and high sensitivity to H2O2, have attracted great attention and are widely used as the protecting or trigger groups in various small molecules or polymer based H2O2-specific fluorescent probes and prodrugs. For example, a bifunctional fluorescent probe mitochondria peroxy yellow 1 (MitoPY1) containing both a peroxide-responsive element (boronate) and a mitochondrial-targeting moiety (triphenylphosphonium headgroup) was reorted.24 This probe performed highly selectively respond to H2O2 over other ROS and showed excellent H2O2 detection ability. However, such small molecule
INTRODUCTION Under the pressure of an increasing numbers of cancerous types and patients, early diagnosis and therapy of cancers have received a growing attention of chemists and biologists. Compared to normal tissues, the abnormalities in tumor sites, such as weak acidity,1−3 unusual temperatures,4,5 hypoxia,6−8 and high levels of reactive small molecules9−11 are always appeared. Although tremendous treatment protocols have been developed, including surgery, radiotherapy, and chemotherapy, etc., conventional treatment mainly relies on small molecule probes and anticancer drugs.12−14 Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) which is the most abundant and stable form of ROS, always act as a messenger in normal cellular signal transduction processes at an appropriate concentration level.15 However, the overproduction of H2O2 will lead to so-called “oxidative stress” and associated with many diseases, including aging, cardiovas© XXXX American Chemical Society
Received: September 22, 2016
A
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Scheme 1. (a) Schematic Illustration for the Fabrication of Oxidation and pH Dual-Responsive Multifunctional CPT@PLLA(IR780)-HBPPI-HPPPI Nanocarriers and (b) Intracellular Milieu-Triggered Programmed Drug Release as Well as ChemoPhotothermol Synergistic Therapy
enhance cellular internalization potency of nanocarriers) were introduced in the nanocarrier design. Moreover, cell-penetrating peptides (CPPs) exhibit a sense of magic; CPPs have excellent membrane permeability and can facilitate intracellular delivery of various cargos (small molecules, macromolecules, and nanoparticles) once integrated with delivery systems. One of the key factors crucial to the high penetration efficiencies of CPPs is their inherent helical structures, especially in the formation of a transmembrane helix.45−47 It would be interesting to integrate helicity into the design of drug delivery systems to potentially develop novel nanovectors with superior membrane permeability. For example, Cheng et al.48 reported the long hydrophobic side chains modified poly(arginine) mimics; they adopt a stable helical conformation and exhibit superior helix-related cell-penetrating properties. Recently, to further extend this hypothesis to total artificial synthetic polymers, we developed a new type of spherical complex micelle with noncharged hydrophilic helical poly(phenyl isocyanide) (PPI) chains stretched out.49 By means of compared experiments, noncharged hydrophilic helical PPIcoated micelles exhibited faster cell membrane permeability than that of normal ones with random linear chain coatings, as evidenced by confocal laser scanning microscopy (CLSM) after incubation of different micelles with HeLa cells at 37 °C. This work demonstrated the importance of helicity on cell membrane permeability and confirmed the helical structure could enhance the biological barriers’ penetration rate, similar to the effect of positively charged CPPs. However, it should be noted that no prospective acceleration was observed; this might be ascribed to the coexistence of right- and left-handed helical conformation on the micellar surface due to the lacking of
probes or drugs are always not only suffered from many obstacles, such as low solubility, poor pharmacokinetics, undesirable biodistribution, inefficient cellular uptake, and an inability to target desired locations but also leading to indiscriminate killing of both cancerous and healthy tissues/ cells.28−33 These undesired side effects may also largely decrease the therapeutic efficacy and outcome. To resolve this problem and optimize therapeutic efficiency as well as reduce side effects, stimuli-responsive polymers are exactly explored to mediate the release profile and perform treatment at desired site.34−38 Responsive polymers can selectively respond to external or endogenous stimuli (e.g., temperature, pH gradient, redox species, enzyme, etc.) through the changes of their chemical or physical properties to adapt to the surrounding environment. Ease of preparation, good biocompatibility, high drug-loading content, excellent stability, and long circulation time endow stimuli-responsive polymeric nanocarriers potential candidates in a series of fields, including bioimaging, drug delivery, theranostic agents, tissue repair materials, etc. However, despite tremendous achievements and progress emerging continuously, the precise control of polymer synthesis, multifunctional integration, on-demand administration, and programmable release of embedded drugs are still difficult challenges. The therapeutic efficacy of traditional nanocarriers is always proved to be less effective because of the existence of several systemic and cellular barriers or the low tissue penetration, leading to low accumulation of nanocarriers and subsequent insufficient drug dose at tumor sites.39 To fill this gap, targeting moieties (folate,40,41 cancer-targeted cRGD,42 and mitochondria-targeted peptides43) and guanidine residues44 (known to B
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Scheme 2. Synthetic Routes Employed for the Successive Copolymerization of L-LA (M1), H2O2-Responsive Phenyl Isocyanide Monomer (M2), and L-Hydrophilic Phenyl Isocyanide Monomer (M3) by Two Mechanistically Distinctive Living Polymerization with ClPd(PEt3)2-IR780-OH as a Single Catalyst To Form PLLA(-IR780)-HBPPI-HPPPI Triblock Copolymers in One Pot
PEGylated single-handed helical HPPPI corona, H2O2responsive HBPPI interlayer, and pH-sensitive PLLA inner core (Scheme 1). Not only that, a layer of NIR absorptive IR780 was anchored at the PLLA−HBPPI interface; the innate NIR absorption character could be served as efficient photothermal conversion and subsequent photothermal therapy medium. The cell-penetrating performance of resultant micelles was first investigated, and the results showed that these micelles coated with hydrophilic single left-handed helical PPI chains exhibited rapid cell membrane permeability and internalization that took only 10−20 min time scale, showing superior features compared with traditional random coil packaged nanovectors. After encapsulants (camptothecin; CPT) loaded, the programmed as well as sustained release could be easily achieved by tuning the H2O2 concentration and solution pH values. The photothermal efficiency was measured by a digital thermometer probe; the hyperthermia induced by the CPT@PLLA(-IR780)-HBPPI-HPPPI micelles could increase to as high as approximately 48 °C upon NIR irradiation (808 nm, 1 W cm−2 ). Combined with the effect of chemotherapeutics, fatal and irreversible damage to cancer cells was observed. The CPT@PLLA(-IR780)-HBPPI-HPPPI micelles were easily taken up by HeLa cells and were primarily located in the acidic organelles after internalization, in favor of pH induced drug release. To the best of our knowledge, although a variety of polymeric nanoparticle-based systems capable of drug delivery and controlled release have been established, nanocarriers surrounded by structural stable hydrophilic single left-handed helical PPI chains exhibiting rapid cell membrane permeability have not been reported. We believe the distinguished micellar structure can serve as a new alternative for nanocarrier design and have potential in practical application.
effective chiral center to induce the single-handed helical conformation, hindering the membrane permeability to a certain extent. In addition to the abnormally high level of H2O2, lower internal pH compared with normal cells is another biological feature of most cancer cells. Therefore, it is interesting to develop dual-responsive polymers that response to both pH and H2O2, especially for the systems that need programmed or synergistically action. Because of its biocompatibility, pH responsiveness, and biodegradability, poly(L-lactic acid) (PLLA)-based materials have attracted considerable investigation on their potential applications as intelligent vectors for gene and anticancer drug delivery.50−52 Herein, motivated by the above designs and existed shortcomings, we herein report the fabrication of oxidation and pH dual-responsive multifunctional polymeric micelles exhibiting rapid cell-penetrating feature, intracellular milieu-triggered programmed drug release, and efficient chemo-photothermal synergistic therapy performance (Scheme 1). Starting from three types of monomers, including two hydrophobic monomers (pH-responsive L-LA monomer, M1; H2O2-responsive phenyl isocyanide monomer, M2) and one hydrophilic phenyl isocyanide monomer (M3) with L-alanine moieties, dual-responsive amphiphilic triblock copolymers, poly(L-lactic acid)(-IR780)-b-hydrophobic poly(phenyl isocyanide)-b-hydrophilic poly(phenyl isocyanide) (PLLA(-IR780)-HBPPI-HPPPI) were synthesized through sequential copolymerization of these three monomers with a near-infrared (NIR) absorptive dye (IR780)-bearing bifunctional catalyst, ClPd(PEt3)2-RhB-OH, by two mechanistically distinctive “one-pot” living polymerization process (Scheme 2). These dual-responsive PLLA(-IR780)-HBPPI-HPPPI triblock copolymers composed of pendent 4-hydroxymethylphenylboronic pinacol ester (4-HPPE) masked carboxyl functionalities could self-assemble into onion-like micelles consisting of C
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Macromolecules Table 1. Molecular Parameters of the Polymers Synthesized in This Work run
[M1]0/[Cat.]0a
[M2]0/[Cat.]0a
[M3]0/[Cat.]0a
Mnb (kDa)
Mw/Mnb
D1:D2:D3c
P1 P2d P3e P4e
30 30
30 30 50
40
30.1 12.8 31.5 22.8
1.17 1.18 1.15 1.14
20:25:35 20:25:0 0:40:35 42:0:35
40 40
50
a
Initial feed ratio of monomers to catalyst. bMn and Mw/Mn values were determined by SEC analyses with equivalent to polystyrene standard. cThe degree of polymerization (Dn) for each block calculated from 1H NMR and SEC. dThis sample is isolated from the batch for P1 preparation. eThese two samples were prepared by a one-pot living polymerization of monomers with a reported Pd(II) complex (ClPd(PEt3)2-OH).56
Figure 1. (a) SEC traces obtained for PLLA(-IR780)-HBPPI-HPPPI triblock and PLLA(-IR780)-HBPPI diblock copolymers using THF as eluent. (b) 1H NMR spectrum obtained for PLLA(-IR780)-HBPPI-HPPPI triblock copolymers. (c) CD and UV−vis spectra obtained for PLLA(-IR780)HBPPI-HPPPI in THF at 25 °C. (d) AFM image obtained for PLLA(-IR780)-HBPPI-HPPPI dried from THF solution (0.2 g/L).
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RESULTS AND DISCUSSION The reliable fabrication of multifunctional nanocarriers through controlled self-assembly of dual-responsive block copolymers with required constitutional units is a prerequisite. We started with the preparation of two kinds of functional monomers, including H2O2-responsive and L-hydrophilic phenyl isocyanide (PI) monomers. For the former, it was synthesized from the exchange reaction between pentafluorophenyl (PFP) esterfunctionalized PI monomers (Figure S1) and 4-hydroxymethylphenylboronic pinacol ester, affording to H2O2-reactive capping moiety and caged carboxyl contained PI monomers, denoted as M2. For the latter, pentane glycol monomethyl ether functionalized L-alanine was first synthesized; then, after undergoing an exchange reaction with PFP ester-functionalized PI monomers, L-hydrophilic PI monomers, denoted as M3, were obtained. M2 can be rapidly oxidized by H2O2, accompanied by the generation of boronic acid, pinacol, 4-(hydroxymethyl)phenol, and carboxyl-functionalized PI monomer through several sequential steps.21 M3 can offer a hydrophilic single-handed helical chains after polymerization. The employed synthetic
routes of M2 and M3 were shown in Figures S2a and S3a, and the synthetic procedures are described in detail in the Supporting Information Experimental Section. The chemical structures of the intermediates during the sample preparation were verified by 1H NMR spectra (Figure S3b), and the exact structures of the final monomers were confirmed by 1H NMR (Figures S2b and S3b), 13C NMR (Figures S2c and S4), and FT-IR spectrometry (Figure S2d). In the next step, IR780encoded bifunctional catalyst ClPd(PEt3)2-IR780-OH bearing a Pd(II) active center and a hydroxyl group was synthesized by sequential chemical modification according to our previously reported literature.49 The employed synthetic routes and procedures are shown in Figures S5a, S6a, and S7a and described in detail in the Supporting Information Experimental Section. The chemical structures of the intermediates during the catalyst preparation were verified by 1H NMR spectra (Figures S5b and S6b), and the exact structure of the final product was confirmed by 1H NMR (Figure S7b), 13C NMR (Figure S7c), 31P NMR (Figure S7d), and FT-IR spectrometry (Figure S7e). D
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Figure 2. (a) Hydrodynamic diameter distribution of the aqueous dispersion of NR@PLLA(-IR780)-HBPPI-HPPPI micelles at different times in the presence of H2O2 (50 mM) and HCl (pH 5.5). (b) TEM images recorded for the morphology evolution of PLLA(-IR780)-HBPPI-HPPPI micelles in the absence and presence of H2O2 and HCl. (c) Fluorescence emission spectra (λex = 550 nm) recorded for the aqueous dispersion of NR@ PLLA(-IR780)-HBPPI-HPPPI micelles at different times in the presence of H2O2 (50 mM) and HCl (pH 5.5). (d) In-vitro dye release profile of NR@PLLA(-IR780)-HBPPI-HPPPI micelles at varying conditions, as monitored by the fluorescence intensity changes at the wavelength of 625 nm corresponding to NR. 1
The well-defined dual-responsive amphiphilic triblock copolymers, poly(L-lactic acid)(-IR780)-b-hydrophobic poly(phenyl isocyanide)-b-hydrophilic poly(phenyl isocyanide) (PLLA(-IR780)-HBPPI-HPPPI), with controlled molecular weights (MWs) and tunable compositions were prepared through sequential living copolymerization in one pot.53−55 The detailed synthetic procedure is illustrated in Scheme 2. Typically, M1 (L-LA; [M]0/[Cat.]0 = 30) and M2 (HBPI; [M]0/ [Cat.]0 = 30) were first copolymerized in the presence of ClPd(PEt3)2-IR780-OH (Cat.) and 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) in 1,2-dichloroethane. After the MW of this diblock copolymer (PLLA(-IR780)-HBPPI; P2 in Table 1) ceased to increase (Mn = 12.8 kDa, Mw/Mn = 1.18; monitored by SEC), degassed M3 (HPPI; [M]0/[Cat.]0 = 40) in 1,2dichloroethane was then added to the reaction mixture via a double-tipped needle. The copolymerization was continued as the second-stage polymerization proceeded. After the copolymerization was completely finished, the chemical structure of isolated PLLA(-IR780)-HBPPI-HPPPI triblock copolymers (P1 in Table 1) was also analyzed by SEC (Figure 1a). The SEC trace of this triblock copolymers exhibited a monomodal elution peak with a Mn of 30.1 kDa and narrow Mw/Mn = 1.17. The actual degree of polymerization (DP) and chemical structure of PLLA(-IR780)-HBPPI-HPPPI were then characterized by 1H NMR analysis (Figure 1b). The spectrum obtained at room temperature revealed that the proton signals attributable to each block could be clearly observed. The UV− vis spectrum also verified the successful preparation of corresponding copolymers (Figure S8). For control experiments, the other two samples, HBPPI-HPPPI (Mn = 31.5 kDa, Mw/Mn = 1.15; P3 in Table 1) and PLLA-HPPPI (Mn = 22.8 kDa, Mw/Mn = 1.14; P4 in Table 1), were also prepared by a reported Pd(II) complex (ClPd(PEt3)2-OH).56 The SEC and
H NMR analysis are shown in Figure S9. All the detailed molecular parameters are summarized in Table 1. The copolymerization of two PI monomers should give optically active block copolymers. From circular dichroism (CD) spectrum (Figure 1c), the negative Cotton effect at 364 nm ascribed to the PPI main chain was clearly observed. The Δε364 of PLLA(-IR780)-HBPPI-HPPPI was estimated to be about −15.6, which indicated that a single left-handed helical chain was formed through the induction of the chiral pendants. Furthermore, the visualized assembly morphology was obtained by tapping-mode atomic force microscope (AFM) technique, as shown in Figure 1d. Spin-coating of a THF solution (0.2 g/L) of PLLA(-IR780)-HBPPI-HPPPI onto a precleaned silicon wafers revealed well-defined nanofibrils with ca. ∼ 60−100 nm diameters and a persistence length of dozens of micrometers were formed. After a careful comparison, the copolymers were found to self-assemble into single left-handed helical fibrils. This result should be resulted from the introduction of Lalanine contained hydrophilic PPI chains and their chiral induction ability during the solvent evaporating assembly process. Given the dual-responsive feature of PLLA(-IR780)-HBPPIHPPPI copolymers, we began with the investigation of H2O2 and pH responsiveness of two control samples (P3 and P4 in Table 1) to establish the corresponding relationship. It is welldocumented that amphiphilic and double hydrophilic block copolymers can self-assemble into numerous morphologies in selective solvents, including spherical micelles, vesicles, and nanorods, depending on the copolymer compositions and cosolvent used.57−60 For both of P3 and P4, spherical nanoparticles were formed in water (Figure S10). Individually, for P3, the hydrodynamic diameter (⟨Dh⟩) of the micelles decreased with incubation time in the presence of various E
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Figure 3. Incubation duration-dependent CLSM images of live HeLa cells when culturing at 37 °C with NR@PLLA(-IR780)-HBPPI-HPPPI micelles. The red channel was excited at 550 nm and collected between 580 and 700 nm.
H2O2 (50 mM) at room temperature, the ⟨Dh⟩ of micelles showed distributions at larger diameter ranges with maximum peak value of ∼95 and ∼110 nm at 3 and 6 h, respectively, indicating the gradually swelling of the micelles. The increase of micelle size should be due to the rapid oxidation of exposed phenylboronic pinacol ester, resulting in the generation of pendent carboxyl groups and the stretching of hydrophobic interlayer. This hydrophobic−hydrophilic transition further increased the polarity of the microenvironment and created interconnected hydrophilic channels in the interlayer, which was beneficial for the interlayer to uptake H2O2 and H2O molecules. With the rapid hydrolysis reaction, more hydrophilic channels in the interlayer were generated, which could not only enhance the chain extension of the interlayer but also promote the subsequent encapsulants release. Further, the acidity of the micelle dispersion was adjusted to pH 5.5, after an additional 2 h incubation, a bimodal rather than unimodal distribution of the ⟨Dh⟩ was observed, presumably due to the formation of single polymer chains resulting from the pH-induced hydrolysis of the PLLA cores. After a total 18 h incubation, the micelles with large diameter ranges were almost disappeared, and only its single chains or possible small multimers formed by selfassociation existed, well verified the specific dual-responsiveness. Moreover, from TEM image (Figure 2b1), we could observe that spherical nanoparticles were formed in pure water, and the size distribution was consistent with the results obtained from DLS (Figure 2a). Then, after H2O2 treatment, the visible micelle size in the TEM image was smaller than that of original ones (Figure 2b2), which was exactly opposite to the DLS results. This conflict should be ascribed to the chain extension of hydrophobic interlayer, leading to the poor contrast in TEM image compared with the compact PLLA core. Furthermore, with synergistic effect of H2O2 and HCl (pH 5.5), TEM image in Figure 2b3 showed the amorphous morphology with slight dark color that was always not visible. This morphology evolution was well consistent with the ⟨Dh⟩
concentrations of H2O2 at room temperature, and higher H2O2 concentration led to faster micelle dissociation (Figures S11a). However, no significant change in ⟨Dh⟩ was observed for the P4 micelles incubated with 100 mM H2O2 for 4 h (Figures S11b), and there was no compromise in the stability of as-assembled micelles after incubation. Thus, it is reasonable to consider that H2O2 play a key role in the size change and confirm that the extreme oxidation sensitivity of phenylboronic pinacol ester to H 2 O 2 . For P4, nile red (NR), a hydrophobic and solvatochromic dye whose fluorescence quantum yield decreases with the increase in polarity of its microenvironment, is used as a fluorescent probe. The pH-responsive NR@PLLAHPPPI micelles as an intelligent drug delivery vehicle were investigated, and the intracellular pH gradient was employed as the triggering event to regulate the release of NR. As expected, NR could be embedded into the hydrophobic PLLA core. The pH-induced release of NR was monitored by the change of the fluorescence intensities recorded for NR (Figure S12). Under weak acidic conditions (pH 5.0 and 6.0), the fluorescence of NR experienced obvious drops (Figures S12a and S12b); however, the fluorescence intensities underwent negligible changes upon being incubated at pH 7.4 (Figure S12c), manifesting the release of NR and the release rates were highly pH-dependent (Figure S12d). Identically, using a cosolvent approach, the amphiphilic copolymers and NR molecules were found to self-assemble into stable aggregates in aqueous solution, leading to the formation of NR@PLLA(-IR780)-HBPPI-HPPPI complex micelles. The dual-responsiveness regulated self-assembled dimension and morphology of NR@PLLA(-IR780)-HBPPI-HPPPI micelles in solution and in the solid state were investigated with dynamic light scattering (DLS) and transmission electron microscope (TEM). As shown in Figure 2a, the NR@PLLA(-IR780)HBPPI-HPPPI micelles showed a symmetric and single model trace with an average diameter to be ∼84 nm (black line), implying the formation of stable nanoaggregates in water. Upon incubating NR@PLLA(-IR780)-HBPPI-HPPPI micelles with F
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Figure 4. (a) UV−vis spectrum and (b) hydrodynamic diameter distribution recorded for the aqueous dispersion of CPT@PLLA(-IR780)-HBPPIHPPPI micelles at pH 7.4; the inset in (b) shows the morphology of micelles observed by SEM. (c) Temperature change of the micellar dispersion (pH 7.4) and PBS as a function of laser irradiation (808 nm, 1.0 W cm−2) time; the inset shows the in-vitro NIR thermal imagings obtained for the micelles (0.5 g/L) and PBS treated by laser for 8 min. (d) Viability of HeLa cells treated with (+; for 8 min) or without (−) laser irradiation and H2O2 after being incubated with various micelles and concentrations. The error bars are based on the standard deviations of four parallel samples.
benefiting from the single left-handed PPI chains had an order of magnitude improvement compared to the conventional nanocarriers and helical chains decorated micelles as reported by our previous literature.49,53 As an effective artificial helical polymers, this distinct feature of single left-handed helical PPI not only plays key roles in their sophisticated functions, such as chiral recognition,61,62 but also opens up a new avenue for exploring next-generation block copolymer-based drug delivery systems with improved efficacy. Subsequently, we engineered the dual-responsive micelles as an intelligent drug delivery carrier, and the intracellular function was also systematically explored. As expected, hydrophobic CPT could be embedded into the hydrophobic region of the micelles, leading to the formation of CPT@PLLA(-IR780)HBPPI-HPPPI micelles. The absorbance spectrum of resultant micelles was used for qualitative analysis, as shown in Figure 4a. Typically, the CPT@PLLA(-IR780)-HBPPI-HPPPI micelles exhibited a strong absorbance at 354 nm for CPT and 800 nm for IR780, indicating the successful loading of anticancer drugs in the micelles. After adjusting the dosage of CPT and PLLA(IR780)-HBPPI-HPPPI, the micelles with a CPT/polymer ratio of 1/5 exhibited the highest encapsulation efficiency (∼74.55% w/w) and loading content (∼6.88% w/w) and were therefore employed in the following experiments. DLS (Figure 4b) showed a symmetric and single model trace with an average diameter of ∼90 nm, implying the formation of stable nanoaggregates in water by the solvent-displacement method; no significant size change or precipitation occurred within 5 days. A scanning electron microscope (SEM) also demonstrated spherical nanoparticles with matched size of DLS. Because of the inherent NIR absorbing ability of IR780, upon NIR laser irradiation, the energy of the laser can be efficiently converted to heat, endowing it to be a photothermal converting agent in photothermal therapy (PTT). To evaluate the
change, reflecting the generation of single polymer chains due to the dual-triggered micelle dissolution. The quantitative relationship between the released NR and treating time was then examined. In neutral environment (pH 7.4), the percent of NR released from NR@PLLA(-IR780)HBPPI-HPPPI micelles was approximately 36% after 8 h incubation with 50 mM H2O2, and a local plateau was observed thereafter, indicating the partial release of loaded NR from interlayer (Figure S13). However, once the pH value of the micelle dispersion was adjusted to 5.5, the NR@PLLA(IR780)-HBPPI-HPPPI micelles showed enhanced NR release up to as high as approximately 62% with an extra 10 h incubation with the coexistence of H2O2 and HCl (Figure 2c). The detailed drug releasing rate and dosage are summarized in Figure 2d; this triggering dependent release manner was favorable for programmed controlling on drug release and alleviating the premature drug release induced systemic cytotoxicity for practical use. As mentioned above, CPPs and poly(arginine) mimics can facilitate cell-penetrating and enhance the delivery efficiency. Herein, the cytotoxic behavior of corresponding PLLA(IR780)-HBPPI-HPPPI micelles was then studied for HeLa cells with a micelle concentration of 0.4 g/L, and the cell viability assays revealed almost noncytotoxic at current concentration. By introducing the hydrophilic single lefthanded helical PPI coronas, the cell membrane permeability and intracellular drug delivery performance based on these dual-responsive NR@PLLA(-IR780)-HBPPI-HPPPI micelles were studied in the next part of work and observed by a confocal laser scanning microscope (CLSM). Surprisingly, upon coincubated with HeLa cells at 37 °C, the intracellular red emission could be observed indistinctly only after 10 min incubation and tended to much brighter and clearer at 20 min (Figure 3). This superfast cell-membrane penetrating rate G
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Figure 5. CLSM images of HeLa cells after coincubating with the aqueous dispersion of CPT@PLLA(-IR780)-HBPPI-HPPPI micelles for 20 and 40 min. Late endosomes and lysosomes were stained with LysoTracker Red (red). The blue channel was excited at 405 nm and collected between 410 and 500 nm.
fluorescence intensity and localization of blue-emitting CPT emission channel. Late endosomes and lysosomes were stained with LysoTracker Red (Figure 5). Upon coincubation with CPT@PLLA(-IR780)-HBPPI-HPPPI micelles for 20 min, effective cellular uptake into HeLa cells was observed. Extending the incubation time to 40 min, cellular uptake to a higher extent was observed with stronger blue emission colocalized with Lyso-Tracker Red, suggesting that these micelles were mainly retained within acidic endolysosomes. Moreover, keeping the same time scale, the superfast cellmembrane penetrating rate brought from distinct single lefthanded helical PPI chains was once again evidenced.
photothermal efficiency, the temperature changes of CPT@ PLLA(-IR780)-HBPPI-HPPPI micelles and PBS (control sample) were monitored in vitro under laser irradiation (808 nm, 1 W/cm2) for 8 min (Figure 4c). The temperature of PBS increased by only approximately 2−3 °C in this case; however, the hyperthermia induced by the CPT@PLLA(-IR780)HBPPI-HPPPI micelles increased to as high as approximately 48 °C, which could lead to fatal and irreversible damage to cancer cells. In the next step, the dose-dependent cytotoxicity of PLLA(IR780)-HBPPI-HPPPI and CPT@PLLA(-IR780)-HBPPIHPPPI micelles was systematically investigated by MTT assay. After incubating HeLa cells with different concentrations of micelles, Figure 4d clearly shows that PLLA(-IR780)HBPPI-HPPPI micelles had negligible cytotoxicity without NIR irradiation (−), independent of the concentration in the range of 0.1−0.4 g/L. However, under laser irradiation (+), the micelles exhibited a remarkable photothermal effect and resulted in a noteworthy reduction in cell viability, especially at high micelle concentrations. Furthermore, the stimulitriggered drug release (chemotherapy) as well as PTT synergistic effect was performed. Apparently, CPT@PLLA(IR780)-HBPPI-HPPPI micelles showed much more cytotoxicity in the assistance of NIR laser, similar to that of PLLA(IR780)-HBPPI-HPPPI ones, but the cell viability did not low enough. Nevertheless, upon incubation the HeLa cells with phorbol 12-myristate-13-acetate (PMA, 0.2 mM; stimulate the secretion of H2O2 by live cell, H2O2(+)) for 5 h, an extra boost in cell toxicity was observed. This dramatic increase of cell toxicity should be due to the chemo-photothermal synergistic therapy that the HBPPI interlayer was first subjected to hydrolysis upon exposure to the generated H2O2 in live cell, giving rise to the increased hydrophily of interlayer and a legitimate release of loaded CPT. Next, the PLLA core experiencing intracellular pH gradient-triggered disintegration occurred naturally, and an enhanced release of CPT was realized, thereby killing cancer cells in a programmed manner. For CPT@PLLA(-IR780)-HBPPI-HPPPI micelles, the cellular uptake kinetics was monitored by the evolution of
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CONCLUSIONS In summary, dual-responsive polymeric micelles fabricated from PLLA(-IR780)-HBPPI-HPPPI triblock copolymers were successfully obtained. The hydrophilic single left-handed helical PPI chains could largely accelerate the cell membrane permeability that only 10−20 min time scale was needed. Moreover, the arylboronate groups contained in the hydrophobic interlayer were stable under neutral and weak acid milieu, and thus could minimize the premature drug leakage and systemic cytotoxicity, but underwent hydrolysis in the presence of H2O2 with extremely high selectivity and specificity and simultaneously generated hydrophilic carboxyl moieties after H2O2 triggered removal of capping moieties. Enhanced drug release was then realized by the introduction of acid milieu, leading to the disruption of PLLA core and subsequent drug release. The release rates could be easily tuned by changing the concentration of H2O2 and the acidity, leading to the programmed as well as on-demand drug release. Upon NIR light irradiation, the temperature of resultant micelles could increase to as high as ∼48 °C. Integrated with the function of chemotherapeutics, fatal and irreversible damage to cancer cells was observed. This work opens a new direction toward the fabrication of smart nanocarriers with rapid cell membrane permeability by taking advantage of single-handed helical polymer chains, and the integrated chemo-photothermal H
DOI: 10.1021/acs.macromol.6b02063 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules
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synergistic therapy method can be utilized to further applications.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b02063. Additional 1H NMR, FT-IR, TEM images, and other characterization data (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (J.Y.). *E-mail:
[email protected] (Z.-Q.W.). Author Contributions
Y.C. and Z.-H.Z. contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by National Natural Scientific Foundation of China (51303044 and 51673058) and the Natural Scientific Foundation of Anhui Province (1408085QE80). Z.W. thanks the Thousand Young Talents Program for Financial Support. J.Y. expresses his thanks for Specialized Research Fund for the Doctoral Program of Higher Education (20130111120013).
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DOI: 10.1021/acs.macromol.6b02063 Macromolecules XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.macromol.6b02063 Macromolecules XXXX, XXX, XXX−XXX
