Microenvironment-activated nanoparticles for oxygen self-supplemented photodynamic cancer therapy

Abstract

Tumor hypoxia, as a hallmark of most solid tumors, poses a serious impediment to O₂-dependent anticancer therapies, such as photodynamic therapy (PDT). Although utilizing nanocarriers to load and transport O₂ to tumor tissues has been proved effective, the therapeutic outcomes have been impeded by the low O₂ capacity and limited tumor penetration of the nanocarriers. To address these problems, we incorporated perfluorooctyl moieties into nanocarriers to improve the encapsulation of perfluorooctyl bromide via fluorophilic interactions, leading to elevated O₂ capacity of the nanocarriers. Meanwhile, to enhance the tumor cell penetrating ability as well as reduce reticuloendothelial system recognition, the nanocarrier was further decorated with a cell-penetrating peptide, which was masked with a protecting group via an acid-labile amide bond for prolonged circulation time and acid-activated cell penetration. The in vitro study demonstrated that, apart from remarkably boosting the photocytoxicity of chlorin 6 (Ce6) at a low dosage, the rationally designed O₂@^DANP_Ce6+PFOB could even alleviate the pre-existing tumor hypoxia. After intravenous injection, O₂@^DANP_Ce6+PFOB exhibited significant tumor accumulation and retention, and potent tumor growth inhibition compared to traditional PDT. Overall, the O₂@^DANP_Ce6+PFOB mediated O₂ self-supplemented PDT with tumor acidic microenviornment-activated cell penetration provides a promising strategy in anticancer treatment.

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Introduction

Photodynamic therapy (PDT), a noninvasive and spatiotemporally controlled therapeutic modality, has been approved for clinical use in treating skin, esophageal, and non-small-cell lung cancers.^1–3 PDT utilizes photosensitizers to transfer light energy of a designated wavelength to tumor-dissolved oxygen (O₂) for generating cytotoxic reactive oxygen species (ROS), such as singlet oxygen (¹O₂) to induce cell death. Therefore, sufficient O₂ supply in tumor tissues is essential for continuous ROS generation, which ensures an uncompromised therapeutic outcome. However, as a hallmark of most solid tumors, hypoxia caused by rapid tumor cell proliferation and distorted tumor vasculature prevents PDT from exerting its full photocytotoxicity.^4,5 Moreover, the continuous O₂ depletion during PDT treatment in turn worsens tumor hypoxia, which further impairs the PDT efficacy and aggravates tumor progression and metastasis.^6–8 Therefore, tumor hypoxia as a promising target for improved cancer therapy has been intensively explored.^6,9–11

To oxygenate tumor tissues for improved PDT outcomes, a number of strategies have been developed,¹² including hyperbaric O₂ inhalation,¹³ tumor vascular normalization,^14,15 increased tumor blood perfusion via mild hypothermia effect,^16,17 direct delivery of O₂ by natural O₂ carriers (e.g., red blood cells, hemoglobin),^18–21 and in situ O₂ generation by decomposing endogenous hydrogen peroxide (H₂O₂) and H₂O with catalase or manganese dioxide (MnO₂),^22–26 and C₃N₄ or graphdiyne oxide nanosheets,^16,27,28 respectively. In particular, perfluorocarbons (PFCs), a class of completely fluorinated carbon compounds, have been widely explored as O₂ carriers in treating lung injury, hemorrhage, cerebral hypoxia, and carbon monoxide poisoning, due to their high O₂ solubility, chemical inertness and excellent biocompatibility.^29–31 Recently, the applications of PFC nanodroplets have been extended to alleviate tumor hypoxia and enhance PDT.^32–34 In this regard, increasing the loading efficiency of PFCs could be an effective approach to elevate the overall O₂ capacity, leading to enhanced PDT efficacy.^35–38 A recent study demonstrated that polyesters with perfluorinated groups could improve PFC encapsulation, which can be attributed to the fluorophilic interactions between PFC and the perfluorinated groups.^39,40 Moreover, apart from efficient O₂ capacity, the therapeutic outcome of PDT has been partly limited by tumor penetration of the nanocarriers, in areas where more severe hypoxia exists.⁴¹ Tumor penetrating peptides can mediate the extravasation transport and enhance the penetration of drugs into tumors via a pathway, dubbed the C-end Rule (CendR) pathway.^42–44

Inspired by this, we fabricated an O₂ self-supplemented nanoplatform to enhance PDT efficacy by simultaneously enhancing O₂ capacity and tumor penetration of photosensitizers. In particular, perfluoroalkyl chains were incorporated into the inner wall of the nanoplatforms, in an effort to improve the encapsulation of perfluorooctyl bromide (PFOB) and ultimately O₂ delivery into tumors. After being co-loaded with a photosensitizer chlorin e6 (Ce6) and saturated with O₂, the O₂ self-supplemented nanoplatform could serve as an O₂ reservoir to replenish O₂ consumed during ROS production, eventually leading to alleviated tumor hypoxia and amplified PDT. Moreover, in order to improve the penetration of photosensitizers into the tumor interior, where more severe hypoxia exists, a tumor acidity-triggered TAT peptide-presenting poly(ethylene glycol)-b-poly(d, L-lactide-co-glycolide) (^DAPEG-b-PLGA)⁴⁵ was incorporated into the O₂ self-supplemented nanoplatform (donated as O₂@^DANP_Ce6+PFOB). The protecting group 2,3-dimethylmaleic anhydride (DA) masked the TAT peptide on the surface of O₂@^DANP_Ce6+PFOBvia an acid-labile amide bond for prolonged circulation time and further acid-activated tumor penetration.^45,47

After i.v. injection, the as-prepared O₂@^DANP_Ce6+PFOB could accumulate at the tumor tissues by the enhanced permeability and retention (EPR) effect.^46,47 The acidic tumoral microenvironment would induce the cleavage of the acid-labile linkage and restore the cell-penetrating ability of the TAT peptide on the surface of O₂@^DANP_Ce6+PFOB, resulting in enhanced tumor accumulation and penetration into the tumor interior. The loaded O₂ could gradually release from O₂@^DANP_Ce6+PFOB for efficiently modulating the tumor hypoxia microenvironment, as well as supplying O₂ for ROS generation upon laser irradiation. Therefore, by employing an O₂ self-supplemented nanoplatform with full biocompatibility, we are able to combine this with PDT to realize antitumor therapeutic outcomes, which holds great promise in preventing tumor relapse and to further clinical PDT applications (Scheme 1).

Scheme 1 (A) Schematic illustration of the preparation process of O₂@^DANP_Ce6+PFOB. (B) Schematic illustration of O₂@^DANP_Ce6+PFOB mediated O₂ self-supplemented PDT. The TAT peptide restores its tumor-penetrating ability upon the detachment of the protective DA corona at the tumoral acidic microenvironment. The O₂ consumption is immediately supplemented by the oxygenated PFOB during Ce6 induced PDT, leading to an enhanced PDT.

To boost the encapsulation of PFOB via fluorophilic interactions, we synthesized an amphiphilic diblock copolymer with perfluoroalkyl side chains by reversible addition–fragmentation chain transfer (RAFT) polymerization using a methyl poly(ethylene glycol) with an ending group 4-(4-cyanopentanoic acid) dithiobenzoate (mPEG-CPAD) as the macromolecular chain transfer agent and 2,2′-azobis(2-methylpropionitrile) (AIBN) as the initiator (Scheme S1†). Monomer N-(tert-butoxycarbonyl)-aminoethyl methacrylate (EABoc-MA) was synthesized by coupling N-(tert-butoxycarbonyl) ethanolamine (EABoc) with methacryloyl chloride, and the structure was confirmed by ¹H NMR spectra (Fig. S1 and S2†). The successful synthesis of the copolymer mPEG-b-P(EABoc-MA) was verified by a shorter retention time compared to that of mPEG-CPAD in gel permeation chromatography (GPC) (Fig. S3†), along with the good agreement in chemical shifts of the ¹H NMR spectrum (Fig. S4†). After deprotection of the amino groups, the perfluorooctyl side chains were grafted to the backbone via the amidation reaction to yield the final product mPEG-b-P(EAMA-g-PFOA) (Fig. S5†), which was then characterized by ¹⁹F NMR and Fourier transform infrared spectroscopy (FTIR) (Fig. S6 and S7†). Moreover, in order to potentiate the cell membrane penetrating ability, the nanoparticles were incorporated with TAT peptide end-capped amphiphilic copolymer TAT-PEG-b-PLGA. The amino groups from the lysine residue of the TAT peptide was further protected by 2,3-dimethylmaleic anhydride (DA) via an acid-labile amide bond, denoted as ^DATAT-PEG-b-PLGA. A detailed synthetic and well-characterized procedure can be found in our previous work.⁴⁵

The as-prepared amphiphilic polymers mPEG-b-P(EAMA-g-PFOA) and ^DATAT-PEG-b-PLGA were blended with Ce6 and PFOB via the emulsion–evaporation method to yield ^DANP_Ce6+PFOB, which was then saturated with O₂ to obtain O₂@^DANP_Ce6+PFOB. The dynamic light scattering (DLS) results showed that the hydrodynamic diameter of ^DANP_Ce6+PFOB increased by approximately 20 nm after O₂ loading (O₂@^DANP_Ce6+PFOB), suggesting its high O₂ capacity (Fig. 1A). Cryogenic transmission electron microscopy (Cryo-TEM) observations revealed that both ^DANP_Ce6+PFOB and O₂@^DANP_Ce6+PFOB have well-defined spherical morphologies,³⁹ with a size and polydispersity in agreement with DLS measurements (Fig. S8†). According to our design, the amide bond between DA and the TAT peptide could be cleaved at mild acidic pH, which can be attributed to the intramolecular nucleophilic attack of the carbonyl group on amides by the neighboring carboxylate group, resulting in the exposure of the amino group in the TAT peptide.⁴⁵ To test the acid-sensitivity of the amide bond, ^DANP_Ce6+PFOB and ^DANP_Ce6+PFOB were incubated in phosphate buffers at pH 6.5 or 7.4 for 4 h, respectively, and the zeta potential changes of NPs were recorded at preset time points. As shown in Fig. 1B, the zeta potential increased from ∼−16.8 mV to ∼0 mV for NPs incubated at pH 6.5, whereas only slight increases were observed when they were incubated at pH 7.4, which could be attributed to the recovery of the positively charged amino groups under acidic pH. Besides, ^DANP_Ce6+PFOB and ^DANP_Ce6+PFOB exhibited great stability at different pH values up to 48 h, indicating that the DA detachment had a minimal effect on the stability of the nanoparticles (Fig. 1C).

Fig. 1 Characterization of O₂@^DANP_Ce6+PFOB. (A) Size distributions of ^DANP_Ce6+PFOB and O₂@^DANP_Ce6+PFOB. (B) The zeta potentials as a function of the incubation time of ^DANP_Ce6+PFOB and O₂@^DANP_Ce6+PFOB in PB buffers (pH 7.4 or 6.5) at 37 °C, respectively. (C) Hydrodynamic diameter changes of ^DANP_Ce6+PFOB and O₂@^DANP_Ce6+PFOB incubated in 10% FBS solution at different pH values. Data are presented as the mean ± S.D. (n = 3).

The characteristic peaks overlapped at 404 nm in the UV-Vis absorbance spectra, indicating the successful loading of Ce6 into ^DANP_Ce6+PFOB, and the drug loading content of Ce6 was 4.87 ± 0.32% (Fig. 2A). Due to the presence of favorable fluorophilic interactions between PFOB and the perfluoroalkyl segments of mPEG-b-P(EAMA-g-PFOA), the encapsulation efficiency of PFOB in ^DANP_Ce6+PFOB was determined to be ∼50%. We then investigated the O₂ capability of ^DANP_Ce6+PFOB in deoxygenated water (pretreated by bubbling N₂ to eliminate O₂) via the Foxy Fospor-R O₂ sensor. O₂@^DANP_Ce6+PFOB exhibited an efficient O₂ loading as well as a gradual O₂ release under hypoxia conditions, as evidenced by a dramatic increase of O₂ concentration in deoxygenated water (Fig. 2B).

Fig. 2 (A) UV-Vis absorbance of free Ce6 and ^DANP_Ce6+PFOB. (B) O₂ release profiles of O₂@^DANP_Ce6+PFOB. (C) The absorbance decay profiles of 9,10-anthracenediyl-bis(methylene) dimalonic acid (ABDA, ¹O₂ probe) at 358 nm in different formulations with or without laser irradiation.

Then, we investigated whether the O₂-enriched O₂@^DANP_Ce6+PFOB would potentiate ROS generation upon laser irradiation using 9,10-anthracenediyl-bis(methylene) dimalonic acid (ABDA) as an indicator. As expected, a sharp decline in the absorbance of ABDA was observed for O₂@^DANP_Ce6+PFOB, indicating its superior ROS generating ability compared to free Ce6 and ^DANP_Ce6+PFOB (traditional PDT regent), which is attributed to the continuous O₂ supplementation during the ROS producing process (Fig. 2C).

On the basis of our previous results that the TAT peptide shielded by DA should be exposed to enhance the internalization of the nano-formulations,⁴⁵ we next examined the acid-activated cell penetrating ability of ^DANP_Ce6+PFOB and O₂@^DANP_Ce6+PFOB by assessing the cellular uptake using confocal laser scanning microscopy (CLSM) (Fig. 3A) and flow cytometry (Fig. 3B) in vitro, respectively. To this end, murine breast cancer 4T1 cells were incubated with different nano-formulations in acidic (pH 6.5) or neutral (pH 7.4) medium for 4 h, and then visualized with CLSM after fluorescent staining. The intracellular fluorescence of cells treated with ^DANP_Ce6+PFOB and O₂@^DANP_Ce6+PFOB in phosphate buffer (pH 6.5) was much stronger than in phosphate buffer (pH 7.4). In particular, the fluorescence intensity of ^DANP_Ce6+PFOB was 23.4-fold higher at pH 6.5 than at pH 7.4, as determined by the quantitative analysis of flow cytometry. The enhanced cellular uptake at pH 6.5 could be attributed to the DA detachment and recovery of the cell-penetrating ability of the TAT peptide at pH 6.5, which in turn facilitated the cell internalization of the TAT-decorated nano-formulations. Moreover, comparable cellular uptakes were observed for ^DANP_Ce6+PFOB and O₂@^DANP_Ce6+PFOB at different pH values, indicating that O₂ loading had a minimal impact on the internalization of the nano-formulations.

Fig. 3 Cellular uptake of O₂@^DANP_Ce6+PFOB. (A and B) Confocal laser scanning microscopy (CLSM) images (A) and FACS results (B) showing the cellular uptake of different formulations in 4T1 cells after incubating in buffers with different pH values (6.5 or 7.4) (scale bar, 20 μm).

Increased cellular internalization of O₂@^DANP_Ce6+PFOB at pH 6.5 should be accompanied by increased intracellular O₂ concentration and the enhanced ROS level upon laser irradiation. We next evaluated the ability of O₂@^DANP_Ce6+PFOB in intracellular hypoxia alleviation and ROS generation. In particular, 4T1 cells were incubated with different formulations (pH 6.5) in a hypoxia chamber (with 1% O₂, 5% CO₂ and 94% N₂), which mimicked the hypoxia microenvironment in tumors. After being subjected to 660 nm laser irradiation, the cells were stained with a hypoxia probe (pimonidazole hydrochloride) and a ROS probe (dichlorodihydrofluorescein diacetate, DCFH-DA) to indicate hypoxia and ROS regions, respectively (Fig. 4). Compared to PBS treated cells, a sharp decrease in the hypoxia fluorescence signal was observed for cells treated with O₂@^DANP_Ce6+PFOB, indicating the excellent O₂ capacity of O₂@^DANP_Ce6+PFOB. In contrast, a significantly enhanced hypoxia fluorescence signal was observed for the free Ce6 and ^DANP_Ce6+PFOB treated cell plus laser irradiation group compared to the PBS group, revealing that O₂ was the pivotal limitation factor in PDT. It is noteworthy that the O₂@^DANP_Ce6+PFOB treated cells, even with laser irradiation, exhibited relatively dim hypoxia signals in comparison with the PBS group. The results indicated that apart from compensating for the O₂ consumption in the production of ROS, the O₂-enriched O₂@^DANP_Ce6+PFOB could further alleviate the pre-existing hypoxia. In line with the hypoxia results, the strongest ROS signal was observed in O₂@^DANP_Ce6+PFOB treated cells, since sufficient O₂ supplementation in O₂@^DANP_Ce6+PFOB continuously fueled the generation of ROS under laser irradiation.

Fig. 4 CLSM images of the intracellular hypoxia and ROS level of 4T1 cells after pre-incubation with different formulations (an equivalent Ce6 dose of 5 μg mL⁻¹) with or without laser irradiation (660 nm, 5 min, 0.5 W cm⁻²) at pH 6.5 under hypoxia conditions (scale bar, 30 μm).

The PDT induced cytotoxicity of O₂@^DANP_Ce6+PFOB was measured by MTT assays. As a negative control, O₂@^DANP_Ce6+PFOB without laser irradiation showed negligible cytotoxicity with the Ce6 concentration even up to 10 μg mL⁻¹, demonstrating excellent biocompatibility in potential applications in the clinic (Fig. 5A). The cytotoxicity of free Ce6, ^DANP_Ce6+PFOB and O₂ self-supplemented O₂@^DANP_Ce6+PFOB exhibited a significant Ce6 concentration in a dose-dependent manner. Notably, the O₂@^DANP_Ce6+PFOB mediated PDT showed a higher cytotoxicity than that of free Ce6 and ^DANP_Ce6+PFOB at lower Ce6 concentrations (e.g. 5 μg mL⁻¹), suggesting the distinct superiority of O₂@^DANP_Ce6+PFOB in PDT therapy. This efficient cytotoxicity of O₂@^DANP_Ce6+PFOB with laser irradiation was further confirmed by live (green fluorescence)/dead (red fluorescence) assays (Fig. 5B). The results suggested that the O₂ self-supplemented strategy could reduce the dose of Ce6 and the possible side effects without compromising PDT efficacy.

Fig. 5 In vitro cytotoxicity examined by MTT assays (A) and Calcein-AM/PI staining (B) in 4T1 cells after pre-incubation with different formulations (at an equivalent Ce6 dose of 5 μg mL⁻¹) with or without laser irradiation (660 nm, 0.5 W cm⁻², 5 min) at pH 6.5 (scale bar, 100 μm).

Intrigued by the superior PDT efficacy of O₂@^DANP_Ce6+PFOBin vitro, we explored the biodistribution and tumor accumulation of O₂@^DANP labeled with a fluorescent dye DiD using the Xenogen IVIS Lumina system. As shown in Fig. 6A and B, the accumulation of O₂@^DANP in tumors gradually increased and the highest fluorescence intensity was observed at 24 h post-injection, and sustained at high levels for up to 48 h. Consistent with real-time whole body fluorescence imaging, the ex vivo distribution investigation of major organs (heart, liver, spleen, lungs and kidneys) and tumors collected 48 h post-injection (Fig. 6C and D) further confirmed the prominent tumor-specific accumulation of O₂@^DANP. The distribution of O₂@^DANP indicated that O₂@^DANP could efficiently accumulate at the tumor site, which may be attributed to a prolonged circulation time and reduced systemic clearance under DA protection. Once reaching the tumor site, the exposure of the TAT peptide on the surface of O₂@^DANP would further enhance accumulation and extend residence time at the tumor tissues. Consistent with our assumption, after 24 h, i.v. injection with O₂@^DANP_Ce6+PFOB caused a significant enhancement of Ce6 accumulation in tumors and deeper penetration in the tumor mass when compared to injecting free Ce6 alone (Fig. S9†), indicating the superior penetration of O₂@^DANP_Ce6+PFOB into the tumor interior.

Fig. 6 Tumoral accumulation of O₂@^DANP. (A) In vivo fluorescence imaging of 4T1 tumor-bearing mice after i.v. injection with DiD labeled O₂@^DANP. (B) Quantification of the fluorescence intensity in the tumor regions at different time points. (C) Fluorescence images of tumors and major organs collected at 48 h post-injection of DiD labeled O₂@NP. (D) Quantification of the fluorescence intensity of organs and tumors (n = 3).

Moreover, the hypoxia level of tumors after the O₂@^DANP_Ce6+PFOB treatment was detected using the hypoxia sensor pimonidazole hydrochloride. Notably, the tumor treated with O₂@^DANP_Ce6+PFOB plus irradiation showed less hypoxic areas and weaker FITC hypoxia signal intensities (green), compared with those treated with free Ce6 plus irradiation and ^DANP_Ce6+PFOB plus irradiation, under which conditions more severe hypoxic areas remained (Fig. S10†), indicating that tumor hypoxia was significantly alleviated by the O₂ supplied by O₂@^DANP_Ce6+PFOB. The above results confirmed that the O₂ self-supplemented PDT strategy with O₂@^DANP_Ce6+PFOB as the O₂ carrier could be a rather effective method to overcome tumor hypoxia and enhance PDT efficacy.

To evaluate the systemic antitumor therapeutic effects of O₂@^DANP_Ce6+PFOBin vivo, 4T1 tumor-bearing mice were randomly divided into five groups and i.v. injected with (Group I) PBS (−), (Group II) O₂@^DANP_Ce6+PFOB(−), (Group III) Ce6 (+), (Group IV) ^DANP_Ce6+PFOB (+), and (Group V) O₂@^DANP_Ce6+PFOB (+) at an equivalent dose of 2.0 mg Ce6 per kg mouse weight (n = 5 for each group), and were either subjected to (+) or not subjected to (−) laser irradiation (Fig. 7A). As shown in Fig. 7B, the administration of O₂@^DANP_Ce6+PFOB without laser irradiation (Group II) or free Ce6 plus laser irradiation (Group III) failed to control tumor growth. Meanwhile, limited by the hypoxia microenvironment in tumor tissues, PDT mediated by ^DANP_Ce6+PFOB plus laser irradiation (Group IV) only exhibited moderate tumor growth suppression. However, with a single treatment of O₂@^DANP_Ce6+PFOB (+) (Group V), the tumor growth was significantly inhibited (85.0% inhibition of tumor growth). The enhanced PDT efficacy could be attributed to the high O₂ capacity and effective O₂ transport into the tumor cells, which continuously supplemented the O₂ consumption during the PDT treatment. The tumor weight measurements were in good accordance with the tumor volume at day 21, indicating the efficient PDT outcome of O₂@^DANP_Ce6+PFOB (Fig. 7C). Besides, no body weight loss was observed for O₂@^DANP_Ce6+PFOB, indicating excellent biocompatibility (Fig. S11†). Moreover, the improved therapeutic outcomes of O₂@^DANP_Ce6+PFOB were further confirmed by H&E staining of tumor tissues, evidenced by obvious nuclear shrinkage and severe tissue necrosis after O₂@^DANP_Ce6+PFOB (+) treatment (Fig. 7D). In addition, hematoxylin & eosin (H&E) histological analysis of major organs (heart, liver, spleen, lungs and kidneys) after different nanoformulations showed negligible tissue damage compared to the PBS group, indicating excellent biocompatibility for all formulations (Fig. 8). In addition, there were no obvious changes in blood cell counts after the treatment with O₂@^DANP_Ce6+PFOB, confirming its biosafety (Fig. S12†). Collectively, the O₂ self-supplemented PDT strategy exhibited superior antitumor efficacy over traditional PDT with negligible systemic toxicity.

Fig. 7 In vivo potent therapeutic efficacies of O₂@^DANP_Ce6+PFOB in the absence and presence of laser irradiation in 4T1 tumor-bearing mice. (A) Schematic of the therapy schedule for 4T1 tumor-bearing mice. The mice were i.v. administered with different formulations and irradiated with (+) or without (−) a 660 nm laser (0.5 W cm⁻², 10 min) at 24 h post-injection. The tumor size was monitored every three days. (B) Variations of tumor volume in the mice after different treatments. Weight of the excised tumors (C), and H&E staining images of the tumor tissue (D) of 4T1 tumor-bearing mice at 21 days after different treatments.

Fig. 8 H&E staining of the heart, liver, spleen, lungs and kidneys of 4T1 tumor-bearing mice after 21 days of treatment with the five formulations (scale bar: 50 μm).

Conclusions

In summary, we reported an O₂ self-supplemented PDT strategy with acid-activated cell penetrating ability to overcome hypoxia-limited therapeutic efficacy. By integrating with a perfluoroalkyl moiety and end-capping with a TAT peptide, O₂@^DANP_Ce6+PFOB exhibited dramatically improved O₂ capacity and cellular internalization. The in vitro hypoxia and ROS fluorescence imaging demonstrated that in addition to compensating for the O₂ consumption during PDT, O₂@^DANP_Ce6+PFOB could partially relieve intrinsic hypoxia. Moreover, O₂@^DANP_Ce6+PFOB achieved enhanced tumor accumulation and retention, owing to the DA corona protection during circulation and acid-activated tumor penetration. Enhanced tumor growth inhibition compared to traditional PDT was observed in the in vivo antitumor study. Taken together, combining O₂ self-supplemented PDT with acid-activated tumor penetration is a new approach in antitumor PDT.

Conflicts of interest

The authors declare that there are no conflicts of interest for this work.

Acknowledgements

This work was supported by the National Key R&D Program of China (2017YFA0205600), the National Natural Science Foundation of China (51773191, 51633008, 51573176, and 21604079), the Fundamental Research Funds for the Central Universities (WK3520000009 and WK2090050042), the Open Project of Key Laboratory of Biomedical Engineering of Guangdong Province (KLBEMGD201701) and the China Postdoctoral Science Foundation (2019TQ0400). This work was partially carried out at the USTC Center for Micro and Nanoscale Research and Fabrication.

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Footnotes

† Electronic supplementary information (ESI) available: Additional experimental details and data include: the syntheses of monomer N-Boc-aminoethyl methacrylate, and block copolymer mPEG-b-P(EABoc-MA); cell culture; cellular uptake; MTT assay; animals and tumor model; biodistribution and antitumor study of O₂@NP_Ce6+PFOB. ¹H NMR spectra of monomer N-Boc-aminoethyl methacrylate, block copolymer mPEG-b-P(EABoc-MA), ¹⁹F NMR and Fourier transform infrared spectroscopy (FT-IR) analysis of mPEG-b-P(EABoc-MA); body weight variation of tumor bearing mice during antitumor treatment. See DOI: 10.1039/c9bm01537j

‡ These authors contributed equally to this work.

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