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High Co-loading Capacity and StimuliResponsive Release Based on Cascade Reaction of Self-Destructive Polymer for Improved Chemo-Photodynamic Therapy Menglin Wang,† Yinglei Zhai,*,‡ Hao Ye,† Qingzhi Lv,† Bingjun Sun,† Cong Luo,† Qikun Jiang,† Haotian Zhang,§ Youjun Xu,∥ Yongkui Jing,⊥ Leaf Huang,¶ Jin Sun,*,† and Zhonggui He*,† †
Department of Pharmaceutics, Wuya College of Innovation, ‡Department of Biomedical Engineering, School of Medical Devices, School of Life Science and Biopharmaceutics, ∥School of Pharmaceutical Engineering, and Key Laboratory of Structure-Based Drug Design & Discovery (Ministry of Education), and ⊥Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, China ¶ Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States §
S Supporting Information *
ABSTRACT: Photodynamic therapy (PDT) shows a promising synergy with chemotherapy in the therapeutic outcome of malignant cancers. The minimal invasiveness and nonsystemic toxicity are appealing advantages of PDT, but combination with chemotherapy brings in the nonselective toxicity. We designed a polymeric nanoparticle system that contains both a chemotherapeutic agent and a photosensitizer to seek improvement for chemo-photodynamic therapy. First, to address the challenge of efficient co-delivery, polymerconjugated doxorubicin (PEG-PBC-TKDOX) was synthesized to load photosensitizer chlorin e6 (Ce6). Ce6 is retained with DOX by a π−π stacking interaction, with high loading (41.9 wt %) and the optimal nanoparticle size (50 nm). Second, light given in PDT treatment not only excites Ce6 to produce cytotoxic reactive oxygen species (ROS) but also spatiotemporally activates a cascade reaction to release the loaded drugs. Finally, we report a self-destructive polymeric carrier (PEG-PBC-TKDOX) that depolymerizes its backbone to facilitate drug release upon ROS stimulus. This is achieved by grafting the ROS-sensitive pendant thioketal to aliphatic polycarbonate. When DOX is covalently modified to this polymer via thioketal, target specificity is controlled by light, and off-target delivery toxicity is mostly avoided. An oral squamous cell carcinoma that is clinically relevant to PDT was used as the cancer model. We put forward a polymeric system with improved efficiency for chemo-photodynamic therapy and reduced off-target toxicity. KEYWORDS: polycarbonate, ROS-sensitive, biodegradable, self-cleavage, chemo-photodynamic, polymer, depolymerization hotodynamic therapy (PDT) was first proposed over 100 years ago. Increasing research is aimed to use harmless photosensitizers and visible light in PDT to ablate malignant tumors. Combining chemotherapy and photodynamic therapy provides a wealth of opportunities to achieve better outcomes. Apart from direct cytotoxicity of reactive oxygen species (ROS), PDT causes acute inflammation and infiltration of leukocytes to boost an immune response.1,2 Additionally, PDT aids to overcome the multidrug resistance of chemotherapeutic agents.3−5 Thus, a drug delivery system to realize efficient co-delivery of the photo-
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© 2019 American Chemical Society
sensitizer and chemodrug but decrease the off-target toxicity of chemotherapy will have great potential for cancer treatment. Technologies in drug delivery systems (DDS) are widely used to combine PDT with chemotherapy. Unfortunately, the loading content of a biocompatible system is very limited. Doxorubicin is among the most effective chemotherapeutic agents. The planar aromatic chromophore portion of DOX is Received: March 18, 2019 Accepted: June 5, 2019 Published: June 5, 2019 7010
DOI: 10.1021/acsnano.9b02096 ACS Nano 2019, 13, 7010−7023
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Cite This: ACS Nano 2019, 13, 7010−7023
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Figure 1. Schematic drawing of the cascade reaction of self-destructive polymeric nanomicelles.
Figure 2. (a) Synthesis route and (b) 1H NMR characterization of PPEG-PBC, PEG-PBC, PEG-PBC-TK, and PEG-PBC-TKDOX.
reported to provide π−π stacking interactions with conjugated systems (e.g., graphene, conductive polymers, and carbon nanotubes). “Core-match” concept was proposed to improve the drug loading capability of nanoemulsions by modifying the loaded drugs with an analogue molecule.6−8 The drug loading of core-matched nanoparticles is increased due to more favorable interactions with similar molecules anchored to the
cores. Photosensitizers also contain the conjugated ring system to allow for a long-lived triplet state after excitation, which may intrinsically favor the π−π stacking interaction with the DOX core.9,10 Over the past decade, polymer-based nanotechnology has been extensively pursued because it offers promising potential for drug delivery. Versatile polymers are designed to realize 7011
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Figure 3. Changes in chemical structure upon ROS activation. (a) NMR spectrum of degradation of PEG-PBC under the condition of Fenton’s reagent with or without the thioketal linker (TK). (b) NMR spectra of Ce6-loaded PEG-PBC TKDOX micelles after 0, 20, and 40 J/cm2 light irradiation. (c) Degradation curves of PEG-PBC-TKDOX nanomicelles in 1, 10, and 20 mM H2O2 analyzed by GPC. Polymer separation was fulfilled by using a gel permeation column coupled with a DAD detector to record the spectrum of DOX. (d) UV absorption of DOX on the polycarbonate chains. (e) Critical micelle concentration of PEG-PBC, PEG-PBC-TK, and PEG-PBC-TK in 10 mM H2O2.
often been applied to conduct a ROS-sensitive delivery.19 Aliphatic polycarbonate (APC) refers to carbonate linkages (−O−C(O)−O−) with no aromatic insertion. We hypothesize that the introduction of thioketal to the pendant chains of APCs would result in the degradation of the polymer in a ROS stimuli-response manner. To address the challenge of efficient co-delivery for both chemotherapeutic agents and photosensitizers, DOX was conjugated to a self-destructive polymeric carrier through a ROS-sensitive pendant thioketal bond (PEG-PBC-TKDOX). Then Ce6 was loaded through the π−π stacking interaction with DOX. The prepared micelle showed high loading for both drugs. By the introduction of thioketal to the pendant chains of the polymer, a cascade reaction is activated to cleave polymer backbones upon ROS stimulus. ROS generated by photodynamic therapy triggers the degradation of nanocarriers to prompt the release of drugs. Therefore, off-target delivery toxicity would be small because DOX is only released where the light is shone. We expected that this strategy induces an added inhibitory effect on tumor growth with increased efficiency and low toxicity (Figure 1).
drug encapsulation, controlled release, stimuli-responsiveness, and specific targeting.11 However, the majority of polymeric nanomedicines have failed to meet clinical approval due to potential toxicity and low delivery efficiency, among which polymer conjugated drugs have a better retention effect in systemic circulation but are confronted with the difficulty of efficient release. In addition, biostable polymers result in side effects including hematotoxicity, nephrotoxicity, hepatotoxicity, and immunogenicity.12,13 An intelligent and biocompatible system with high drug loading and delivery efficiency is in demand. We hypothesize that carrier degradation sensitively induced by an external stimulus will benefit delivery efficiency and reduce the above-mentioned toxicities. To design a desirable elimination pattern, we focused on chalcogen-mediated cleavage strategies.14−17 Notably for synthetic prodrugs, a sulfhydryl-assisted cleavage strategy is applied to promote the release of the probe drug, as nucleophilic sulfhydryl preferentially attacks the carbonate bond. The β-positioned disulfanyl−ethyl carbonate is the most prevalently used linker, which forms intramolecular pentacyclization after the reductive breakage of the disulfide bond.18 Thioketal, another sulfhydryl-based ROS-responsive bond, has 7012
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Figure 4. (a) Dynamic light scattering size of intensity weighting Gaussian distribution and (b) zeta-potential of Ce6-loaded PEG-PBCTKDOX nanomicelles. (c) Transmission electron microscopy images and appearance of Ce6-loaded PEG-PBC-TKDOX micelles before and after laser irradiation at 660 nm. (d) UV absorption spectrum indicating red shifts in series of the mass ratio of DOX and Ce6 loaded in PEG-PBCTKDOX micelles. (e) Fluorescence spectra of Ce6 and DOX solution, as well as the nanomicelles encapsulating both drugs. An ex 400 nm was set for DOX excitation, and ex 480 nm was for Ce6. (f) Accumulative release of PEG-PBC-TKDOX(Ce6) micelles. The fluorescence of released DOX was quantified. (g) Accumulative release of PEG-PBC-TKDOX(Ce6) micelles. The fluorescence of released Ce6 was quantified. (h) Loading efficiency and particle size corresponding with the increasing loading content of Ce6 in PEG-PBC-TKDOX (n = 3).
alcoholysis.24 The high conjugation rate of thioketal is an advantage for sulfhydryl-mediated degradation. PEG-PBC-TK showed rapid reduction in molecular weight in 10 and 20 mM hydrogen peroxide (H2O2), detected by gel permeation chromatography (GPC) (Figure S10). It is indicated that, upon ROS stimulus, 70.6% of molecular weight was reduced in 1 h (Figure 3c). To clarify the underlying mechanism of polymer degradation, we investigated the necessity of the thioketal (TK) motif in the degradation reaction. The degradation of PPEG-PBC was only initiated by simultaneous addition of both TK (3,3′-(propane-2,2-diylbis(sulfanediyl))dipropanoic acid) and Fenton’s reagent, suggesting that the polycarbonate was potently cleaved by thioketal under ROS conditions in 1 h (Figure 3a). After DOX was grafted to the side chains, the PEG-PBC-TKDOX micelles also displayed a rapid H2O2-responsive reduction in molecular weight, coupled with DOX release (Figure 3c). We used HPLC-DAD, connected to a GPC column, to quantify the grafted DOX on the polymer (Figure 3d). To verify the chemical changes in nanoparticles, we compared the 1H NMR spectra of the
RESULTS AND DISCUSSION Characterization of External Stimulus-Induced SelfDegrading Polymer. Light-guided therapy based on photoactivated carriers is widely pursued.20−22 To address the challenges of suboptimal degradation, we synthesized a selfdestructive aliphatic polycarbonate that cuts itself to lower molecular mass upon ROS activation. This is achieved by grafting the thioketal to the pendant chains of the aliphatic polycarbonate (PEG-PBC-TK). The degree of polymerization was optimized by the drug loading capacity in our previous work.23 To synthesize a ROS-responsive polymer, thioketal linkages were grafted to the pendant hydroxy of polycarbonates (PEG-PBC) via postpolymerization modification (Figure 2). The intermediate molecules were confirmed by both mass and 1 H NMR spectra (Figures S1−S6), as well as by differential scanning calorimetry (Figure S7) and infrared spectroscopy (Figure S8). The degree of substitution can be as high as 77.5% (Figure S9), which was realized by the formation of the corresponding thioketal anhydride followed by an efficient 7013
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ACS Nano Table 1. Characteristics of Ce6-Loaded PEG-PBC, PEG-PBC-TK, and PEG-PBC-TKDOX Micelles polymers
Mn,NMR
Dh (nm)
polydispersity index
drug loading efficiency (%)
drug loading content (%)
PEG-PBC PEG-PBC-TK PEG-PBC-TKDOX
9154 14950 19150
149.7 162.6 51.4
0.268 0.384 0.314
21.7 ± 0.9 20.8 ± 0.6 95.0 ± 5.2
9.8 ± 0.4 9.4 ± 0.2 32.2 ± 1.2
Figure 5. Intracellular distribution of ROS-triggered self-cleavable PEG-PBC-TKDOX. (a) Nucleus distribution of KB cells. The cells are irradiated with a 660 nm laser (40 J/cm2) at 1 h after incubation with drugs and then another 3 h incubation before staining. The bars indicate 20 μm. (b) Colocalization of lysotracker with DOX after PEG-PBC TKDOX(Ce6) and PEG-PBC TKDOX(Ce6)L+ treatment. Images of KB cells treated with Ce6-loaded PEG PBC-TKDOX micelles for 1 h and then laser irradiation. The bars indicate 50 μm. (c) Colocalization of DOX and nuclear stain DAPI were analyzed by ImageJ (n = 3, ****, p < 0.0001). (d) Overlapped pixels of lysotracker (blue) with DOX (red) were quantified by ImageJ (n = 3, **, p < 0.01, ***, p < 0.001). (e) Cell viability detected by MTT assay.
nanoparticles with or without laser irradiation. Significant changes in chemical composition were found when the nanoparticles were exposed to a 660 nm laser (40 J/cm2). The cleavage of thioketal linkages and the destruction of the polycarbonate backbone were supported by evidence of the reduction in peak at δ 1.5 and the simultaneous reduction in peak at δ 3.9 (Figure 3b). When PEG-PBC-TK was subjected to ROS circumstances, critical micelle concentration of the amphiphilic polymer increased from 10−8 to 10−5 g/mL, indicating the dissociation of the polycarbonate segment (Figure 3e). Self-Destructive Nanoparticles for Chemo-Photodynamic Drug Delivery. DOX on the pendant chains served as
a hydrophobic core that compacted the nanoparticles, supported by evidence of the quenching of the fluorescence intensity (Figure 4e). No fluorescence from DOX (ex 480 nm) was recorded due to aggregate formation in the condensed phase, namely, aggregation-caused quenching (ACQ).25 The π−π stacking interactions between Ce6 and DOX extended the conjugated electron, which resulted in a red shift in absorption spectra of the nanoparticles. When the ratio of Ce6 and conjugated DOX reached 1:1, the red shift mostly saturated (Figure 4d). The molar ratio of 1:1 also exhibited the highest loading efficiency among groups, reflecting the highest absorption. We tested the loading capacity of PEG-PBCTKDOX. With the increasing encapsulation of Ce6, the 7014
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Figure 6. Increased tumor accumulation and penetration with photodynamic assistance. (a) Penetration of DOX in MCF7/ADR 3D cell spheroids. Light irradiation was applied at 4 h after drug treatment and followed by another 4 h incubation before imaging. (b) Fluorescence imaging detecting DOX penetration in tumors. The tumors were irradiated with 660 nm laser (40 J/cm2) at 8 h after i.v. injection. Tumor frozen sections were prepared 4 h later and (c) quantitation of DOX accumulation was presented. (e) Accumulation of Ce6 in nude mice at 12 h after i.v. injection. Fluorescence of Ce6 was acquired by ex/em of 630/700 nm, and (d) mean intensity of the region of interest was quantified. (f) DOX in tumors was extracted and measured at 24 h (n = 5, *, p < 0.05, **, p < 0.01 by student’s t test). (g) Pharmacokinetics profiling of DOX concentration in serum versus time. (h) Biodistribution of DOX solution and PEG-PBC-TKDOX(Ce6) micelles at 12 h with or without light irradiation. (i) Biodistribution of Ce6 solution and PEG-PBC-TKDOX(Ce6) micelles at 12 h with or without light irradiation.
out the following experiments using nanoparticles loading 20% Ce6 content with a 21.9% DOX mass ratio (Figure S9). We then compared the loading capacity of Ce6 in PEG-PBC, PEGPBC-TK, and PEG-PBC-TKDOX. The polymer with DOX conjugation showed an increase in drug loading content by 3.4fold with reduced size and size distribution (Table 1). The
particle size of PEG-PBC-TKDOX nanomicelles decreased to 55.0 nm, followed by a sharp increase to 333.9 nm. The polymeric nanomicelle exhibited a high loading capacity up to 50% for Ce6, with loading efficiency greater than 95.0% (Figure 4h). As is reported by Chan et al., 40 and 50 nm nanoparticles demonstrated the greatest effect.26 We carried 7015
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PBC-TKDOX and Ce6 also worked in a strong synergy to induce cytotoxicity of malignant tumors cells (Figures 5e and S16). The proliferation of cells treated with PEG-PBCTKDOX(Ce6)L+ was significantly inhibited, compared with the single treatments. A thorough inhibition was achieved with combinatory treatment using a high dose. Nevertheless, no toxicity to KB cells was observed for the non-DOX-conjugated polymers (PEG-PBC and PEG-PBC-TK), which suggested a safe delivery even at a high dose (500 μg/mL) (Figure S17). Improved Delivery Efficiency in Vivo. We hypothesized that ROS generated by PDT was responsible for the target specificity of PEG-PBC-TKDOX. We cultured 3D cell spheroids to test the permeability of PEG-PBC-TKDOX in vitro. Because the KB cells cannot form cell spheroids in our culture condition, we chose a DOX-resistant breast cancer cell line (MCF7/ADR) as an alternative. The photodynamic effect helps to overcome the drug resistance, which facilitates the deeper penetration by down-regulating expression of an efflux transporter like P-glycoprotein.29−32 It is concluded from the z-stack images of spheroids that the internalization of free DOX was very limited. Polymer-conjugated DOX (PEG-PBCTKDOX) penetrated deeper probably because of the evasion of efflux transporters (e.g., P-glycoprotein). In contrast, addition of light irradiation to PEG-PBC-TKDOX micelles resulted in the most cellular uptake of DOX in spheroids (Figure 6a). We then evaluated the pharmacokinetic property of PEG-PBC-TKDOX(Ce6) by determining the plasma drug concentration. The conjugated polymeric prodrug PEG-PBCTKDOX sufficiently increased the area under the curve (AUC) of DOX by 4.8-fold (Figure 6g). Ce6 showed high affiliation to bull serum albumin (Figure S19), and albumin in serum served as the sink condition for Ce6.33,34 The PK profile of Ce6 was in a different pattern with DOX (Figure S20), which however did not significantly influence the effect of ROS-induced polymer degradation at tumor sites. In vivo with the KB tumor model, accumulation of DOX at 12 h postinjection was determined by confocal imaging and further compared with PEG-PBC-TKDOX treatments. Nanoparticle technology prominently increased the penetration depth, but the addition of light irradiation was critical in elevating the drug accumulation in vivo (Figure 6b,c). The accumulation of Ce6 was significantly increased at 12 h, as quantified by region of interest determination, respectively (Figure 6d,e). The optimal time of irradiation (12 h postinjection) was decided by accumulation of Ce6 imaged by an IVIS system (Figure S18). At 12 h after i.v. injection, the mice were sacrificed, and DOX and Ce6 in major organs were extracted and determined. Two main reasons may result in the different distribution pattern between free DOX and the DOX motif released from the PEG-PBC-TKDOX. On one hand, positively charged DOX preferentially accumulates in the mitochondria of myocytes due its high affinity for a negatively charged lipid abundance in heart tissue, known as cardiolipin.35 After modification via an amide bond, the positively charge amino group of DOX was blocked, which might facilitate the elimination of DOX through the kidneys.36 On the other hand, the DOX derivatives containing a thiol group were cleaved from the PEG-PBC-TKDOX micelles without destruction of the amide when exposed to high ROS.37 The hydrophobic small fractions were predominantly eliminated by the renal system.38 The distribution in tumors at 12 h postinjection (light given at 8 h) for both drugs was significantly elevated by 2.6- and 4.86-fold, respectively (Figure 6h,i). At 24 h, the
DOX conjugation was attributed to the significant size decrease by inserting π−π interactions, which resisted the negative charge of PEG-PBC-TK and Ce6. The loading efficiency of Ce6 in PEG-PBC-TKDOX was as high as 95.0% (Table 1). PEG-PBC had a loading capacity comparable to that of PEG-PBC-TK, indicating that even when the ROSsensitive bond is cleaved, the polymer may not instantly release the loading cargos. More than 8 out of 35 carboxyl groups were conjugated with DOX in a single polymeric molecule (Mn = 19,150.4 Da), calculated from 1H NMR data (Figure S9). PEG-PBC-TKDOX micelles loaded with Ce6 were prepared by a simple solvent evaporation method, with zeta-potential centered at zero and size centered at about 50 nm (Figure 4a,b). Particle morphology imaged by transmission electron microscopy (TEM) showed a round and refined shape of the particles (Figure 4c). Theoretically, the self-cleavable and disassembling strategy enables quick release and confers ontarget distribution of polyprodrug (PEG-PBC-TKDOX). TEM showed the destruction of Ce6-loaded nanoparticles in morphology after laser irradiation. The round particles no longer maintained their original shape and became aggregated after irradiation (40 J/cm2), which is confirmed by changes in particle size determined by dynamic light scattering (Figure S11). The incorporated DOX showed its intrinsic red color after laser irradiation, which potentially indicates the release of DOX (Figure 4c). As a result, the nanoparticles release the conjugated cargos in a ROS-dependent manner (Figure 4f,g). On-Target Intracellular Delivery. Next, we examined the improvement in therapeutic effect in a human oral squamous cell carcinoma cell line (KB). Due to the advantage of local PDT, the PEG-PBC-TKDOX nanoparticle enabled more drug exposure to tumor cells, which is induced by site-specific irradiation. Two main barriers need to be addressed toward the drug−polymer conjugation delivery system. First, nanoparticles are mainly taken up through the endocytosis pathway and end up in endosomes, compromising the efficacy of therapeutic options. The cellular uptake of PEG-PBC-TKDOX(Ce6) micelles can be efficiently inhibited by chlorpromazine (inhibitor of clathrin-dependent endocytosis) (Figure S12). Interestingly, in our research, the overlap of lysosome and the DOX dramatically decreased from 84.5% without irradiation to 46.3% with light irradiation (p < 0.05), indicating the successful endosome escape of nanoparticles (Figure 5b,d). On the other hand, doxorubicin interacts with DNA by stabilizing the topoisomerase II complex, thereby inhibiting the process of DNA replication. When adding laser irradiation, we observed that 83.7% (with light irradiation) versus 37.7% (nonradiation) of DOX overlapped with the cell nucleus at 4 h after incubation with drugs (Figure 5a,c). We found similar results of the nucleus distribution in the MCF-7 cell line (Figure S13). These data suggested that the addition of ROS benefited the DOX distribution to the nucleus, possibly due to more efficient release from the polymer backbone. DOX is reported to transport to the nucleus by forming a DOX− proteasome complex. Without ROS assistance, it is difficult to release the conjugated DOX from the nanoparticles. Therefore, the efficiency of translocation to the nucleus is lower for PEGPBC-TKDOX, thus inhibiting the efficient interaction with DNA or topoisomerase II.27,28 There is no significant difference in intracellular uptake between the DOX and PEG-PBC-TKDOX groups in terms of cellular uptake on KB cells at 3 h, as detected by low cytometry as well as the fluorescence in the cell lysate (Figures S14 and S15). PEG7016
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Figure 7. Enhanced in vivo therapeutic effect and reduced toxicity. (a) Growth curves of subcutaneously inoculated KB tumors. Intravenous injections were given to mice at day 7, day 9, and day 11. The 660 nm laser irradiation followed at 12 h after each injection. (b) Tumor burden is indicated as tumor weight versus body weight at the end point. (c) Body weight changes (n = 5, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001). (d) Tumor images at the end point. (e) Tumor images of H&E staining, Ki67 immunohistochemistry, and TUNEL assay after the treatment depicting morphology, proliferation, and apoptosis, respectively. (f) H&E morphology of main organs at the end of treatment. Arrows highlight the regions of metastasis (blue), congested glomeruli (yellow), and cardiac rhabdomyolysis (black). (g) Serum biochemical marker analysis (n = 3, *, p < 0.05, **, p < 0.01).
known, doxorubicin works as a first-line chemotherapeutic agent, efficiently blocking the proliferation of malignant tumors.44 However, severe weight loss in hosts was found in the DOX group (Figure 7c). Two out of five mice died from the 2 mg/kg of DOX doses. In contrast, no significant weight loss and animal deaths were found in the PEG-PBC-TKDOX group with or without light irradiation (L+). PEG-PBCTKDOX(Ce6)L+ suppressed tumor growth within 14 days, and the average tumor burden in mice was significantly reduced in comparison to that in the other groups (Figure 7a,b,d). Proliferation marker Ki67 diminished after PEG-PBCTKDOX (Ce6)L+ treatment, and TUNEL assay detected the most apoptotic DNA in this group (Figure 7e). Although free DOX suppresses the tumor growth significantly, severe cardiac rhabdomyolysis was found in a pathology examination. In contrast, there was no obvious morphological
concentration of DOX released via light irradiation in the tumors is still 2.2 times higher than that in the nonirradiation group (Figure 6f). Although a nanocarrier enhanced the tumor accumulation by the EPR effect (
