Photosensitive Pro-drug Nanoassemblies Harboring A Chemotherapeutic Dormancy Function Potentiates Cancer Immunotherapy

Abstract

Immunotherapy combined with effective therapeutics such as chemotherapy and photodynamic therapy is a successful strategy to activate anti-tumor immune responses for improved anticancer treatment. However, developing multifunctional biodegradable, biocompatible, low-toxic but highly efficient, and clinically available transformed nano-immunostimulants remains a challenge and is in great demand. Herein, we report and design a novel carrier-free photo-chemotherapeutic nano-prodrug COS-BA/Ce6 NPs by combining three multifunctional components self-assembled natural small molecule betulinic acid (BA), a water-soluble chitosan oligosaccharide (COS), and a low toxic photosensitizer chlorin e6 (Ce6) to augment the antitumor efficacy of the immune adjuvant anti-PD-L1-mediated cancer immunotherapy. We show that the designed nanodrugs harbored a smart and distinctive "dormancy" characteristic in chemotherapeutic effect with desired lower cytotoxicity, and multiple favorable therapeutic features including improved 1 O2 generation induced by the reduced energy gap of Ce6, pH-responsiveness, good biodegradability, and biocompatibility, ensuring a highly efficient, synergistic photochemotherapy. Moreover, when combined with anti-PD-L1 therapy, both nano-coassembly-based chemotherapy and chemotherapy/photodynamic therapy (PDT) could effectively activate antitumor immunity when treating primary or distant tumors, opening up potentially attractive possibilities for clinical immunotherapy.

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KEYWORDS 

Cancer immunotherapy; Nano-immunostimulants; Chemotherapeutic dormancy; Pro-drug; Self-assembly; Natural small molecule; Betulinic acid; Photodynamic therapy

1. Introduction 

Cancer immunotherapy, such as the immune checkpoint blockade strategy using PD-L1 or CTLA-4 to combat cancer by activating the host's immune system, has undoubtedly revolutionized cancer treatment1,2. Unfortunately, the weak immune response and ineffective therapeutic effects greatly limited its clinical prospects3,4. Recently, current studies have revealed that a combination of immunotherapy with other modalities such as chemotherapy5, photodynamic therapy (PDT)6, photothermal therapy (PTT)7, and radiotherapy8, can effectively trigger antitumor immunogenicity by inducing cancer cells to undergo immunogenic cell death (ICD)9, which suggests the potential of immune modulation to activate anti-tumor immune responses. Numerous innovative designs of nanomaterials based on these synergistic combinations of immunotherapy strategies have achieved significantly improved anti-cancer activity including inorganic10, polymer11, and biomimetic12 nanoparticles (NPs). However, developing multifunctional biodegradable, biocompatible, low toxicity but highly efficient, and clinically available transformed nano-immunostimulants remains a great challenge and huge demand. Natural biological materials like nucleic acids13, proteins12, peptides14,15, and small molecular natural products16,17 due to their inherent benefits (e.g., biocompatibility, non-toxicity) have drawn a significant research interest for medical applications. Especially in recent years, anticancer bioactive terpenoid natural small molecules (NSMs) possessing a self-assembly function have been discovered successively18,19. For example, betulinic acid (BA), ergosterol (ET), and abietic acid (AA) can self-assemble into micron or nano-structured particles. These NSMs are attractive building blocks to use to develop antitumor agents for synergistic chemotherapy and PDT. They have been proven to exhibit significant benefits of easy nano-fabrication, better biocompatibility and biodegradability, lower toxicity with reduced liver injury, and high synergism in antitumor therapy20e23. This provides promising alternatives for developing clinically available translatable nano-immunostimulants for synergistic immunotherapy. However, the generally stronger lipophilicity of these NSMs led to inefficient cellular endocytosis, which weakened their anticancer activity and limited their clinical value for cancer treatment24. Similar to the commonly reported nanocomposites, the corresponding NSMs nano-formulations promoted cellular uptake to enhance chemotherapeutic effects, but also inadvertently increased the risk of nonspecific cytotoxicity to normal cells22. Therefore, how to maximize the anticancer efficacy of terpenoid NSM nano-agents while minimizing its toxicity to normal tissue is worth thinking deeply about as a crucial issue in the synergistic immunotherapy process.

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Pro-drug strategies have been extensively applied to improve the cell uptake of lipophilic small molecules by increasing hydrophilicity25. Generally, amphiphilic drugs particularly possess higher cellular uptake efficiency than hydrophilic or lipophilic drugs26. The higher the cellular phagocytosis, the better the therapeutic efficacy24. Thus, developing amphiphilic terpenoid NSMs pro-drug molecules may be a potential method for maximizing their therapeutic effects. In addition, stimuli-responsive nanodrugs to the specific tumor microenvironment (e.g., acid, redox responsiveness) hold great promise for controlling drug release, which is advantageous in suppressing the cytotoxicity of the chemotherapeutic drugs27,28. Meanwhile, supramolecular coassembly (e.g., a reprecipitation approach) was an effective strategy to prepare highly water-soluble carrier-free nanodrugs22,29, which might be expected to slightly decrease the phagocytosis efficiency of amphiphilic NSMs to some degree, thereby attenuating their pre-designed cytotoxicity. We hypothesize that stimuli-responsive and fully hydrophilic nanodrugs were further established based on amphiphilic NSMs pro-drug and can effectively suppress its nonspecific cytotoxicity to normal cells while stimulating the release of highly toxic amphiphilic NSMs into tumor cells, facilitating their dormancy in normal tissues and activation in tumor site. It is important to select the appropriate water-soluble and stimuli-responsive precursor to modify terpenoid NSMs to prepare amphiphilic pro-drug molecules. For years, chitosan oligosaccharides (COS), as the degraded products of chitosan, not only have good water solubility and abundant eOH oreNH2 groups that lead to a good pH response but also have beneficial antitumor activities by enhancing the host's immune function through regulating the activity of immune cells such as monocyte macrophages and lymphocytes30,31. These guarantee the huge possibility of constructing stimuli-responsive target nano immunostimulants for synergistic immunotherapy.

Herein, based on the above considerations, in our experimental design, a pentacyclic triterpene NSMs of betulinic acid (BA) is used as a potent anticancer agent32, COS was introduced to synthesize an amphiphilic COS-BA molecule to improve the chemotherapeutic effect. Chlorin e6 (Ce6), a widely utilized photosensitizer with very low/no dark toxicity33, was employed to construct a completely water-soluble carrier-free photosensitive prodrug COS-BA/Ce6 NPs using a direct co-assembly strategy. COS-BA/Ce6 NPs combined two therapeutic modalities, chemotherapy and photodynamic therapy (PDT), which can effectively increase the presentation of tumor-derived antigens to T cells, generating robust immunological responses with the assistance of an immune adjuvant anti-PD-L1 to cause highly efficient and synergistic anti-tumor chemotherapy/PDT/immunotherapy (Scheme 1). The engineered COS-BA/Ce6 NPs showed a smart dormancy function with insidious chemotherapy effects, i.e., nonirradiated co-assembled NPs showed remarkably inhibited in vitro cytotoxicity compared to amphiphilic COS-BA whether against cancer or normal cells. Attributing to the distinctive pH-responsive behavior, it performs low toxicity and biosafety to normal tissue in vivo but is highly toxic to tumor tissue due to the stimulating release of hyperactive COS-BA in the weakly acidic tumor microenvironment34. To our best knowledge, currently, the designed nano drugs harboring this chemotherapeutic dormancy function are still rarely reported. Meanwhile, the nanodrug has multiple favorable therapeutic features such as improved 1 O2 generation by reducing the energy gap (DEST) of Ce6, rapid biological metabolism, and enhanced tumor accumulation, which led to an efficient and safe antitumor combination of chemotherapy and PDT treatment. More importantly, both NP-based chemotherapy and chemotherapy/PDT systems could effectively activate antitumor immunity by facilitating the proliferation of TNF-a and IFN-g-producing CD8þ T cells in primary or distant tumors. Our proposed biodegradable, biocompatible, uniquely dormant, bio-safe, and highly efficient photo-chemotherapeutic nano-drug achieves significant systemic immune therapeutic responses, opening up attractive possibilities for clinical application in treating distant or metastatic cancer therapy.

Scheme 1 PDT and chemotherapy of co-assembled COS-BA/Ce6 NPs potentiated anti-PD-L1 to induce a systemic antitumor immunotherapy. The introduction of water-soluble COS can effectively enhance the chemotherapeutic activity of BA (depicted as a half-sword), resulting in a hyperactive COS-BA amphiphilic prodrug (long-sword). When co-assembled with photosensitizer Ce6 (scabbard), the shell-structured and pH-sensitive nano-assemblies had a smart and distinctive "dormancy" function with inhibited chemotherapeutic toxicity to normal tissues. But, once it arrived and was stimulated by the acidic (Hþ) tumor microenvironment, the highly active amphiphilic prodrug COS-BA will be released resulting in significant chemotherapy. In addition, the constructed co-assemblies exhibited excellent PDT activity with enhanced singlet oxygen ( 1 O2) generation induced by the reduced energy gap between the singlet and triplet excited states (DEST) of Ce6. Combined with an anti-PD-L1 checkpoint blockade, the release of tumor-associated antigens after chemotherapy/PDT treatment can effectively activate a systemic antitumor immune response for distant cancer immunotherapy.

2. Materials and methods 

2.1. Synthesis of COS-BA 

Betulinic acid (40 mg, 0.1 mmol), EDC (38 mg, 0.2 mmol), and NHS (17 mg, 0.15 mmol) were dissolved in 3 mL of N, N-dimethylformamide (DMF). After stirring at room temperature for 30 min, 96 mg of COS water solution (1 mL) was gently added dropwise into the mixture and stirred for 24 h. Then the reaction mixture was diluted with CH2Cl2, and extracted repeatedly. The CH2Cl2 layer was evaporated and purified on a silica gel column. The unreacted BA was removed initially using petroleum ether: acetone (5:1, v/v) as eluent, and then changed to 3:1 to yield white COS-BA (yield: 42%), where COS was identified as dimer (n Z 2). 1 H NMR (400 MHz, CDCl3) d: 8.98 (1H, s, BA-28 O] CeNH), 4.73 (1H, s, H-29a), 4.60 (1H, s, H-29b), 3.18 (1H, dd, J Z 4.8, 10.8 Hz, H-3), 2.95e3.93 (8H, m, COS), 2.64 (3H, s, CH3eCOS), 0.76 (3H, s, H-24), 0.83 (3H, s, H-25), 0.90 (3H, s, H-23), 0.97 (3H, s, H-26), 0.98 (3H, s, H-27), 1.70 (3H, s, H-30). 13C NMR (100 MHz, CDCl3) d: 179.18 (CO, COS), 156.43 (C- 28), 150.97 (C-20), 109.03 (C-29), 78.74, 77.04, 69.32, 69.22, 65.25, 58.14, 55.62, 55.21, 53.65, 53.22, 50.52, 45.96, 44.32,44.11, 41.85, 40.59, 38.68, 38.55, 37.32, 37.04, 36.39, 35.31, 34.21, 31.81, 31.54, 31.17, 30.28, 29.09, 27.80, 27.22, 25.74, 25.53, 20.91, 19.48, 18.12, 16.02, 15.87, 15.17, 14.65, 14.62. ESIeMS m/z: 814.5871 [M‒6H] (C44H72N2O12). 

2.2. Preparation of COS-BA/Ce6 NPs 

Co-assembled COS-BA/Ce6 NPs were prepared by one step reprecipitation method. Typically, 5 mL of Ce6 (33 mmol/L) and 40 mL of COS-BA (33 mmol/L) DMSO solutions were first mixed. Subsequently, the mixture was injected rapidly into 1 mL of water containing 10 mL of NaOH (50 mmol/L) under ultrasonication (KQ-250E, ShuMei, Kunshan, China). After sonication for 10 min, the COS-BA/Ce6 NPs were obtained by centrifugation at 13,000 rpm (TGL-16G, Xiangyi, Changsha, China) for 25 min. Other formulations with different ratios of COS-BA to Ce6 were prepared by the same procedure with a constant NaOH to Ce6 molar ratio of 3:1. 

2.3. Cell lines 

Human MCF-7, HepG2, LO2, and mouse 4T1, L929 cell lines are provided by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). MCF-7, HepG2, and LO2 were cultured in DMEM medium with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin antibiotic (Life Technology, USA) at 37  C. 4T1 and L929 were cultured in RMPI-1640 medium with the same ratio as above. 

2.4. Cellular uptake and endocytic pathways studies 

For investigation of cellular uptake, 4T1 or LO2 cells were incubated with free Ce6 or COS-BA/Ce6 NPs (equivalent concentration of Ce6: 1.7 mg/mL, COS-BA: 19.5 mg/mL, BA: 10.8 mg/mL) for various times. After washing, fixing, and DIPA (#D9542, Sigma) staining, the cells were imaged under a fluorescent inverted microscope (FIM) (DSY-2000X, UP Optotech, Changchun, China). The fluorescence intensity was determined using flow cytometry using the Guava EasyCyte mini system (Merck Millipore, Germany). For quantification of the uptake efficiency, the cellular accumulation of BA and COS-BA was measured using HPLC (Agilent 1200, USA). Typically, 4T1 cells were treated with free COS-BA or BA (equivalent BA: 25 mg/mL) for 0.5 and 3 h. After washing with PBS, the cell pellets were harvested and underwent ultrasonic homogenization (JY92-IIN, Scientz Biotechnology, Ningbo, China) for cell lysis. Then, COS-BA or BA was extracted by chloroform for HPLC analysis after lyophilizing the samples. The mobile phase was water (0.2% phosphoric acid) and acetonitrile at a flow rate of 1.0 mL/min, and corresponding volume ratios were 60:40 and 15:85 (v/v) for analysis of COS-BA and BA, respectively. The detection wavelength was 213 nm. For investigation of endocytic pathways of COS-BA/Ce6 NPs, 4T1 cells were seeded in 6-well plates and incubated for 24 h before being pretreated with various endocytosis inhibitor for 30 min, including a micropinocytosis inhibitor [50 mmol/L, ethyl isopropyl amiloride (EIPA) (#N132356, Aladdin)], a clathrin-mediated endocytosis inhibitor [20 mmol/L, chlorpromazine (#C7010, Solarbio)], a caveolae-mediated endocytosis inhibitor [25 mmol/L, nystatin (#IN0260, Solarbio)], and a lysosome acidification inhibitor [50 mmol/L, chloroquine phosphate (#C9720, Solarbio)]35. Next, the cells were further cultured with 2 mL of COS-BA/Ce6 NPs (Ce6: 1.7 mg/mL, COS-BA: 19.5 mg/mL) for 2 h and imaged under FIM (UP Optotech). The cells incubated without endocytic inhibitor at 37  C were set as control and at low-temperature incubation (4  C) for evaluation of the energy-dependent endocytosis. Meanwhile, the mean fluorescence intensity of cells was also determined by flow cytometry. 

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2.5. Cytotoxicity assay 

4T1 cells were seeded in a 96-well plate. Then the cells were treated with various concentrations of COS-BA NPs for 24 h. Afterward, 10 mL of MTT (#M158055, Aladdin) dye (5 mg/mL) was added to the well and incubated for an additional 4 h, and then 150 mL of DMSO was added to dissolve the formazan crystals. The absorbance of each well at 492 nm was recorded by a microplate reader. For the irradiation group, the cells were treated with drugs for 4 h, then irradiated for 10 min by 675  10 nm light and incubated for an additional 20 h. The cytotoxicity of COS-BA/ Ce6 NPs (1 mg/mL of Ce6 corresponds to 11.5 mg/mL COS-BA and 6.4 mg/mL BA) against other cells (MCF-7, HepG2, LO2, and L929) were also determined by the same MTT assay only with a different medium. Meanwhile, the combination index was calculated by the software CompuSyn 2.0 using Chou Talalay theorem calculation36.

2.6. Animal models 

All experimental procedures were executed according to the protocols approved by the Harbin Medical University Animal Care and Use Committee. 4T1 cells were suspended in RMPI-1640 medium and subcutaneously injected to the right-back of each female BALB/c mouse (18e22 g, 6e7 weeks old). 

2.7. In/ex vivo fluorescence imaging 

A 200 mL of COS-BA/Ce6 NPs 5% glucose solution (equivalent Ce6: 0.35 mg/mL, COS-BA: 4.02 mg/mL) was injected through the tail vein into tumor-bearing mice. After that, fluorescence imaging of tumor sites at a predetermined time was performed using an AniView 100/600 multi-model in vivo animal imaging system (Guangzhou Biolight Biotechnology Co., Ltd., China) with the excitation at 630 nm. After 24 h post-injection, the major organs (heart, liver, spleen, lung, and kidney) and tumor were collected for ex vivo fluorescence imaging. Besides, the ex vivo fluorescence images of tumors and major organs at different times after i.v. injections of Ce6 or COS-BA/Ce6 NPs were also evaluated. 

2.8. In vivo chemotherapy/PDT 

4T1-bearing mice (75e100 mm3 ) are randomly divided into six groups (n Z 5): 1) 5% glucose solution; 2) Ce6; 3) Ce6 with irradiation; 4) COS-BA (40.2 mg/kg, where BA: 22.3 mg/kg); 5) COS-BA/Ce6 NPs; 6) COS-BA/Ce6 NPs with irradiation. For each group, mice are injected with 200 mL of COS-BA/Ce6 NPs (equivalent Ce6: 3.5 mg/kg of the body) through the tail vein on Days 0, 2, and 4. For the irradiation groups, at 4 h post-injection after each administration, all mice were anesthetized with isoflurane, and the tumor sites were irradiated by 675  10 nm (150 mW/cm2 ) light for 15 min. Tumor volumes and body weights of mice are recorded every other day as well. Tumor volumes are calculated by the formula: V Z width2  length/2. After 14 days of treatment, the tumors were excised and weighted to calculate the tumor inhibition ratio (TIR) by the commonly used equation: TIRZ (1 sample/control)  100%. Where sample and control were the average tumor weight of each treated group and 5% glucose aqueous solution group, respectively. 

2.9. In vivo chemotherapy/PDT/immunotherapy 

To demonstrate the potential of COS-BA/Ce6 NPs combined with immunotherapy, the bilateral tumor model was designed through subcutaneous injection of 4T1 cells into the left and right flanks. When the tumors approached 75e100 mm3, the mice were divided into five groups. (1) Control; (2) COS-BA/ Ce6 NPs (COS-BA Z 40.2 mg/kg, where BA Z 22.3 mg/kg); (3) COS-BA/Ce6 NPs þ light; (4) COS-BA/Ce6 NPs þ anti-PDL1; (5) COS-BA/Ce6 NPs þ anti-PD-L1 þ light. On Day 0, 200 mL of 5% glucose solution (Group 1) or COS-BA/Ce6 NPs (Ce6 Z 3.5 mg/kg) (Group 2, 3, 4, 5) were i.v. injected into mice. After 4 h post-injection, the right tumors of Groups 3 and 4 were irradiated for 15 min. The mice were anesthetized with isoflurane and rolled on one side to avoid exposing the left tumors. Then anti-PD-L1 antibody (BioXcell, product number: BE0101, clone number: 10F.9G2) was i.v. injected into mice for Groups 4 and 5 on Days 2, 4, and 6 (15 mg per mouse per injection). The length and width of each tumor were monitored every other day. After 14 days of treatment, the blood serum of mice was collected to evaluate interferon-gamma (IFN-g) (#KMC4021) and tumor necrosis factor (TNF-a) (#BMS607-3) using ELISA assay (eBioscience). The tumors were collected, dissociated, treated with red blood cell lysis buffer, and washed with PBS by centrifugation. Then, the cell suspension was stained with anti-CD3-FITC (#MA1-10187, clone 145-2C11, eBioscience) and anti-CD8a-PE (#12-0081-85, clone 53-6.7, eBioscience). Afterward, the cytotoxic T lymphocyte (CTL) infiltration in the right and left tumors after different treatments were evaluated by flow cytometry.

3. Results and discussion 

3.1. Synthesis, fabrication, and characterization of COS-BA/ Ce6 NPs

In this study, we design a novel prodrug small molecule COS-BA based on water-soluble chitosan oligosaccharide (COS), which may elevate the chemotherapeutic activity of pentacyclic triterpene betulinic acid (BA) by improving the cell uptake through increasing hydrophilicity. The COS (degree of polymerization, DP: 2e6) employed here was prepared by our previously described method30. Then, the COS-BA was synthesized via a dehydration condensation reaction. Combined with the principal component analysis of COS, the chemical structure of COS-BA was thought to be a disaccharide (DP:2) modified BA (Supporting Information Fig. S1), which was confirmed by 1 H NMR, 13C NMR, and ESIeMS (Supporting Information Figs. S2eS4). Analysis of the representative 1 H NMR showed the distinct proton signal of COS around 2.64e3.93 ppm and C]CH2 peaks (4.60, 4.73 ppm) of BA, demonstrating the successful synthesis of COS-BA (Fig. 1A). For preparation of the coassembled synergistic antitumor reagent COS-BA/Ce6 NPs, a versatile re-precipitation approach was employed by cooperating with a highly efficient photosensitizer Ce6. By changing the initial molar ratios, both COS-BA/Ce6 co-assembly formulations could form spherical nanostructures with modulated sizes (Fig. 1B and Supporting Information Fig. S5). Across different input ratios of COS-BA/Ce6, the hydrophilic diameters of COS-BA/Ce6 NPs were near 200e300 nm with a negative z-potential. Considering that NPs prepared at a formulation of 8:1 showed a lower polydispersity index (PDI, 0.143) with a relatively smaller size (249 nm), a sufficient amount of chemotherapeutic agent COS-BA was also required for the combinatorial treatment. Therefore, we chose the formulations of COS-BA/Ce6 (8:1) for the following study. Under this optimization, COS-BA/Ce6 NPs exhibited narrow size distributions (Fig. 1C) and good water-solubility, indicating its colloidal nature (Fig. 1D). To explore the reasons behind this successful co-assembly, the primary self-assembly behavior of free COS-BA was investigated using scanning electron microscopy (SEM) (Fig. 1E), which suggested that COS-BA could form a highly uniform nano-sphere of w168 nm, implying that the chemical modification of COS does not alter the self-assembly ability of BA itself (Supporting Information Fig. S6). It is that property that eventually induces the successful construction of the co-assemblies. Notably, apart from the solid nanostructure confirmed by transmission electron microscopy (TEM), COS-BA/ Ce6 NPs showed a distinct shell structure free COS-BA NPs, and the average shell thickness was approximately 2.35 nm. The other physiochemical characterization is summarized in Fig. 1F. Besides, COS-BA/Ce6 NPs showed a negative z-potential of  5.38 mV and hydrodynamic size of w249 nm, higher than that obtained from SEM (w182 nm). Simultaneously, Ce6 loading and encapsulation efficiencies were 8.1 and 96%, respectively, as determined by high-performance liquid chromatography (HPLC). Also, FT-IR data (Fig. 1G) further confirmed this co-assembly because COS-BA/Ce6 NPs exhibited the characteristic C]N, CeN, and C]CH peaks of Ce6 appearing at 1633, 1078, and 633 cm 1, respectively. 

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3.2. Co-assembled mechanism and the formation of the shell structure

Subsequently, to better comprehend the co-assembly mechanism, we first investigated the primary formation process of NPs. We found that the co-assembly of COS-BA with Ce6 may be spontaneous because these two species could also form spherical nanostructure even if without sonication (Supporting Information Fig. S7), which may be due to the negative Gibbs free energy change (DG ) in co-assemblies37. This spontaneous assembly also suggested a stronger intermolecular interaction between COS-BA and Ce6. Generally, the intermolecular interactions in supramolecular self (or co-) assemblies are noncovalent p‒p-stacking, hydrogen bonding, and van der Waals forces 38,39. For this analysis, we then investigated the UV absorption of COS-BA/Ce6 NPs (Fig. 1H and F), which suggested a red-shifted Soret (410 nm) and Qy (667 nm) bands compared to free Ce6 (405 and 665 nm, respectively). Meanwhile, the significant red-shifted characteristic absorption of COS-BA can also be found, indicating a possible intermolecular p-p stacking between COS-BA and Ce640. Moreover, after incubating with the surfactant sodium dodecyl sulfate (SDS, 0.2% w/v) (Supporting Information Fig. S8), COS-BA/Ce6 NPs showed a newly blue-shifted Qy band at 641 nm and dramatically recovered fluorescence, demonstrating that the hydrophobic interactions also contributed to the co-assembly28. Since these associative noncovalent interactions, co-assemblies showed remarkable fluorescence quenching (98.2%) in water (Fig. 1I). Collectively, it is reasonable to believe that p‒p stacking and hydrophobic interactions may be the main driving forces for the construction of co-assemblies. Afterward, to further visualize the assembly process more intuitively and reveal the underlying reasons why shell-structured NPs can form, we performed a simplified molecular dynamics (MD) simulation model containing 16 optimized COS-BA molecules and an additional two Ce6 in a water box. Initially, we evaluated the possible interaction/binding of COS-BA self-assemblies within 5 ns of simulation time (Fig. 2A and Supporting Information Fig. S9). We found that, except for numerous intramolecular hydrogen bonds in the COS fraction, several pairs of intermolecular H-bonds between COS-BA could also be observed (Fig. 2B and Supporting Information Fig. S10), which indicated that hydrogen bonding may be one of the main driving forces for the formation of COS-BA NPs. Particularly interesting, in COS-BA self-assemblies, all COS-BA molecules form an apparently symmetrical structure (Fig. 2C). This fascinating phenomenon motivated us to explore the possible relationship between this property and the formation of shell-structured COSBA NPs. Then, we attempted to expand the units of the box. Surprisingly, the connected boxes consisting of COS-BA molecules were arranged in a regular manner resulting in an "orthogonal" hollow network structures (Supporting Information Fig. S11). More importantly, we randomly calculated the thickness of the shell molecules and found that were consistent with the TEM (w2.35 nm) (Fig. 2D). This tendency to form hollow network structures may be responsible for the formation of shellstructured NPs. To test this idea, the MD structure of COS-BA/ Ce6 co-assemblies was simulated (Fig. 2E and Supporting Information Fig. S12). Similar to COS-BA self-assemblies, a large number of intramolecular and intermolecular H-bonds also were found in the co-assemblies (Fig. 2F and Supporting Information Fig. S13). Additionally, the possible p-p stacking between C]C of COS-BA and the aromatic ring of Ce6 was observed in the co-assemblies (Fig. 2G), which is consistent with the data from previous UV investigations. After extending the number of boxes, similar hollow network structures can still be found, only with reduced regularity and orthogonality (Supporting Information Fig. S14). This may be the possible reason for the non-uniform morphology of COS-BA/Ce6 NPs compared to COSBA NPs. Moreover, the molecular shell-layer distance is also close to the shell thickness (w2.35 nm) of NPs as seen using the TEM (Fig. 2H). Collectively, we speculated that the formation of shell structured COS-BA/Ce6 NPs or COS-BA NPs might largely be attributable to this tendency of molecules to assemble into hollow shell network structures.

Figure 1 Fabrication and characterization of chitosan oligosaccharides-modified betulinic acid (COS-BA) prodrug-mediated nano-assemblies COS-BA/Ce6 NPs. (A) The amplified key positions of 1 H NMR of COS-BA. (B) Preparation of co-assembled COS-BA/Ce6 NPs with various COS-BA/Ce6 ratios. (C) Size distribution of COS-BA/Ce6 NPs using COS-BA and Ce6 (molar ratio, 8:1) as building blocks. (D) The water solubility and Tyndall effect of co-assemblies, indicate a colloidal nature. (E) SEM and TEM images of free COS-BA self-assemblies and co-assembled COS-BA/Ce6 NPs. (F) Summary of physiochemical properties of COS-BA/Ce6 NPs. (G) FT-IR spectra, (H) UV Vis spectra, and (I) fluorescence emission spectra of free Ce6, COS-BA, and co-assembled COS-BA/Ce6 NPs in water.

Figure 2 Structures of (A) COS-BA self-assemblies and (E) co-assembled COS-BA/Ce6 NPs after molecular dynamics (MD) simulation for 5 ns with a structural unit molar ratio of 8:1 (COS-BA/Ce6, calculated based on Ce6 loading efficiency). The line model was used for H2O and the stick models represent COS-BA or Ce6 molecules, where the C atoms of Ce6 are labeled with purple. The hydrogen bond information between COS-BA molecules in (B) its self-assemblies and (F) COS-BA/Ce6 NPs co-assemblies. (C) The apparently symmetrical structure of the COS-BA molecular arrangement after MD simulation. (G) Possible molecular interactions between COS-BA and Ce6 show potential p-p stacking. The indicated thickness of the shell molecules in an "orthogonal" hollow network structure after extending corresponding MD boxes of (D) free COSBA self-assemblies and (H) COS-BA/Ce6 NPs co-assemblies.

3.3. Energy mechanism of enhanced 1 O2 generation 

Next, we further examined the generation of singlet oxygen (1 O2), one of the critical reactive oxygen species (ROS) of PDT performance using DPBF (1,3-diphenylisobenzofuran) as a probe. After sustained irradiation of the mixture solutions of COS-BA/ Ce6 NPs with DPBF, the UV absorption of DPBF decreased in intensity, indicating 1 O2 generation (Fig. 3A). After comparison with the methylene blue (MB) reference, the F ( 1 O2) of COS-BA/ Ce6 NPs was calculated as 0.26, slightly higher than free Ce6 (FZ0.19) (Fig. 3B). Generally, the energy absorbed by photosensitizers will undergo three relaxation pathways involving fluorescence, heating effect via vibrational relaxation, and ROS generation through an intersystem crossing (ISC)41. Consistent with the significantly quenched fluorescence emission, COS-BA/ Ce6 NPs also did not promote any heating effect in water (Supporting Information Fig. S15), which implied an enhanced ISC process after co-assembly of Ce6 with COS-BA, thus leading to the improved 1 O2 generation. To further elucidate the molecular mechanism of such an effect, we attempted to optimize and calculate the possible single-molecule model of COS-BA/Ce6 consisting of potential p-p stacking obtained from MD simulation by density functional theory (DFT) (Fig. 3C). Amazingly, COS-BA/Ce6 showed an observable red-shifted UV absorption of 581.24 nm than that of free Ce6 (579.30 nm), indicating the occurrence of p-p stacking (Fig. 3D). Although the energy minimized geometry of COS-BA/Ce6 showed slight structural differences with inputted MD configuration, the distinctive pstacking between C]C or C]O bonds of COS-BA with the porphine ring of Ce6 was still observable, further validating that the above p‒p stacking is possible. Meanwhile, it has been recently demonstrated that the increased electron-donating efficiency by p‒p stacking could facilitate the ISC process42, while the ISC rate (KISC) is proportional to (1/DEST) 2,43. That is, the lower energy gap (DEST) of photosensitizer will enhance the ISC process promoting efficient ROS generation. We subsequently calculate the lowest excited singlet state (S1) and the triplet state (T1) of Ce6 and COS-BA/Ce6 (Fig. 3E), respectively. The results indicated that COS-BA/Ce6 showed a decreased DEST of 1.1892 eV compared to free Ce6 (1.2001 eV), confirming the enhanced ISC process. The reduction of DEST is helpful for 1 O2 production, which has also been verified in other nanophotosensitizers44. Taken together, the p-stacking seems to reduce the DEST of Ce6 after co-assembly with COS-BA, thus leading to improved 1 O2 generation.

Figure 3 Energy mechanism of improved 1 O2 generation induced by COS-BA/Ce6 NPs. (A) UV Vis spectra of photodecomposition of 1,3-diphenylisobenzofuran (DPBF) after prolonged irradiation of COS-BA/Ce6 NPs. (B) In the first-order plots of DPBF UV absorption changes (DOD), the 1 O2 yield was evaluated using methylene blue (MB) (F Z 0.52) in DMSO as the reference compound. (C) Energy-minimized structures of a possible single-molecule COS-BA/Ce6 model viewed from the side and front, which was obtained according to the initial molecular configuration between Ce6 with COS-BA after MD simulation and following DFT calculations. (D) Predicted UV Vis absorption spectra transitions (vertical bars) of free Ce6 and an optimized COS-BA/Ce6 model. (E) Calculated energy levels of their lowest excited singlet state (S1) and lowest triplet state (T1), respectively, [Gaussian 09/B3LYP/6-31G (d, p)]. Where DEST was calculated as ES1  ET1.

3.4. Chemotherapy dormancy characteristics 

Afterward, to validate the design concept in the present study that developing amphiphilic COS-BA molecule could effectively increase the therapeutic effect of BA by improving the cell uptake through increasing hydrophilicity, and meanwhile further constructing stimuli-responsive and fully hydrophilic COS-BA/Ce6 NPs facilitated its dormancy in normal tissues and activation in tumor sites. To test this hypothesis, we first investigated the drug release behavior of COS-BA/Ce6 NPs in various buffer solutions (Fig. 4A). The results indicated that COS-BA/Ce6 NPs showed a pH-responsive behavior with a cumulative Ce6 release of 88% at pH 5.6 and 57% at pH 7.4 after 24 h, respectively. This feature likely arose from the destruction of numerous hydrogen bonds that existed in COS-BA/Ce6 NPs after the introduction of acidic Hþ45, thus leading to increased Ce6 release (Fig. 4B). This was further confirmed by the disassembly of COS-BA/Ce6 NPs after incubation in acidic environments directly (Fig. 4C). This pH-responsive feature may be beneficial for an effective antitumor therapy due to the weakly acidic tumor microenvironment34. Meanwhile, COS-BA/Ce6 NPs had better stability because no significant size or PDI changes could be observed after incubation in PBS and 1640-RMPI cell culture medium for 10 days (Supporting Information Fig. S16), which would be important for their potential use for further biomedical applications. Subsequently, further investigations were conducted by evaluating the cytotoxicity of free lipophilic BA (contact angle q: 133.3 ) and amphiphilic COS-BA (q: 52 ) (Fig. 4D) by a standard methyl thiazolyl-diphenyl tetrazolium bromide (MTT) assays. As predicted, either for 4T1 cancer cells or normal LO2 hepatocytes, individual COS has essentially no anticancer activity at relatively low doses. This might be due to the anti-proliferation effects of COS being activated via inducing NF-kB signaling pathway only at high concentrations (approximately at milligram level)30. Whereas, COS-BA performs remarkably enhanced cytotoxicity with extremely low IC50 values (6.03 mg/mL for 4T1 and 9.92 mg/mL for LO2) compared to those of BA (17.63 mg/mL for 4T1 and 20.53 mg/mL for LO2) even at the same mass concentrations (Fig. 4E and J). The elicited results implied that modification of water-soluble COS greatly enhances the cellular uptake efficiency of lipophilic BA, thus leading to the strong anticancer activity of COS-BA. To quantify the uptake efficiency, the accumulation of BA or COS-BA within 4T1 cells was directly determined by HPLC (Supporting Information Fig. S17). The results clearly supported that amphiphilic COS-BA exhibits a significantly higher uptake than free BA (approximately 3.6-fold after 3 h of incubation), demonstrating the enhanced chemotherapeutic activity of COS-BA. The above conclusion was further supported by the significantly improved cytotoxicity of amphiphilic COSBA against HepG2 and MCF-7 cancer cells (Supporting Information Fig. S18), which are well consistent with the design concept. After co-assembly with Ce6, we found that COS-BA/Ce6 NPs without irradiation exhibit remarkably inhibited cytotoxicity against 4T1 cancer cells compared to the equivalent COS-BA, especially at 11.5 mg/mL, but the basic PDT activity was not affected (Fig. 4F). To validate this phenomenon more fully, we further investigated the in vitro cytotoxicity of COS-BA/Ce6 NPs against other cancer cells including MCF-7 and HepG2. Interestingly, amphiphilic COS-BA showed very stronger cytotoxicity against MCF-7 cells (Fig. 4G), but the chemotherapeutic activity decreased significantly after co-assembly with Ce6, i.e., nonirradiated COS-BA/Ce6 NPs. Similar results were also observed in HepG2 cells only with a slight decrease (Fig. 4H). To further support the above conclusions, we also evaluated the chemotherapeutic effects of nano-assemblies and COS-BA against normal mouse fibroblast L929 cells and human LO2 hepatocytes (Fig. 4I). The results indicated that the assembled COS-BA/Ce6 NPs significantly reduced the original highly toxic amphiphilic drug COS-BA, showing excellent biocompatibility. By analyzing the cytotoxicity, the IC50 values of the amphiphilic COS-BA were significantly lower than that of the co-assembled COS-BA/Ce6 NPs regardless of cancer or normal cells (Fig. 4J). That is to say, constructing completely hydrophilic and stimuli-responsive nano coassemblies can effectively suppress the high cytotoxicity of amphiphilic drug COS-BA in advance. Considering the distinctive pH-responsive feature, it seems that COS-BA/Ce6 NPs will be dormant in normal tissues with desired low chemotherapy toxicity, but active in tumor tissues owing to their stimulating release of highly toxic COS-BA in the weakly acidic tumor microenvironment. Taken together, unlike the widely reported neofunctionalization of chemotherapeutic reagents that have enhanced anticancer activity22,23, amphiphilic COS-BA mediated nano-assemblies not only exhibit a smart "dormancy" characteristic with insidious chemotherapy effects but also retain specialized PDT activity, showing huge potential for smart antitumor therapy.

Figure 4 (A) In vitro Ce6 release profiles from COS-BA/Ce6 NPs in PBS (pH 7.4, 6.5 or 5.6) containing Tween 80 (0.5%, v/v) at 37  C. (B) The possible pH-responsive mechanism that Hþ triggers the disassembly of NPs. (C) The disassembly of COS-BA/Ce6 NPs after direct incubation in approximately pH 4.0 water. (D) The contact angle of free BA, Ce6, COS-BA, and COS-BA/Ce6 NPs, respectively. (E) Cell cytotoxicity of free BA, COS, and amphiphilic COS-BA against representative cancer 4T1 and normal LO2 cells. Cell viability of (F) 4T1, (G) MCF-7, (H) HepG2 cancer cells after being incubated with free COS-BA, COS-BA/Ce6 NPs with/without irradiation at indicated equivalent COS-BA concentration for 24 h. (I) Cell cytotoxicity of free COS-BA, non-irradiated COS-BA/Ce6 NPs against normal LO2 and L929 cells. (J) The IC50 analysis of COS-BA against cancer and normal cells. Data are expressed as means  SD (n Z 6). *P ˂ 0.05, **P ˂ 0.01, and ***P ˂ 0.001 show the significant differences.

To explore the possible reasons behind this chemotherapy dormancy characteristic, the time-dependent cellular uptake behavior of COS-BA/Ce6 NPs was evaluated successively by incubating with 4T1 cancer cells. The results indicated that, although COS-BA/Ce6 NPs showed remarkable fluorescence quenching, they still showed enhanced phagocytic efficiency compared to free Ce6 owing to the stronger cytoplasmic red fluorescence, as determined by flow cytometry quantitative analysis (Fig. 5A). This improved cellular internalization of NPs may be ascribed to endocytosis21. Taken together, we speculated that, although highly water-soluble co-assemblies COS-BA/Ce6 NPs (contact angle q: 36.7 ) exhibited improved cellular endocytosis than lipophilic Ce6 (q: 132.6, close to BA), the reduced phagocytosis efficiency of NPs might appear compared to amphiphilic COS-BA molecule, thus appearing suppressed the chemotherapeutic cytotoxicity of COS-BA/Ce6 NPs. That is to say, the cell uptake efficiency from high to low as such sequence: amphiphilic COS-BA, hydrophilic COS-BA/Ce6 NPs, lipophilic BA or Ce6. More importantly, the inadequate disassembly of cellular NPs may be another important reason for the weakened chemotherapeutic effect, all of which may be related to the smart chemotherapy dormancy of COS-BA/Ce6 NPs. Besides, because the disassembly of NPs was specifically conducted in the acidic environment of tumor cells, to demonstrate the pH-responsive feature, we further attempted to compare the uptake effects of COS-BA/Ce6 NPs in normal LO2 cells (Fig. 5B). However, COS-BA/Ce6 NPs did not exhibit significantly inhibited Ce6 red fluorescence in LO2 cells. In contrast, the uptake behavior by LO2 was similar to that of 4T1 cancer cells, suggesting that COS-BA/Ce6 NPs can also disassemble even in normal cells, which may result from the weakly acidic pH intracellular compartments (endosomes/lysosomes) existing inside both cancer and normal cells46. Since the cellular uptake of NPs depends on the process of endocytosis, the direct drug incubation might not highlight the pH-responsiveness of COS-BA/Ce6 NPs at the cellular level. Nevertheless, the tumor microenvironment (TME) is highly acidic compared to normal tissues47. Regardless of cancer intracellular or TME, COS-BA/ Ce6 NPs can be pH-responsive and disassembled to trigger the release of highly toxic COS-BA. Whereas, in normal tissues, COS-BA/Ce6 NPs could only be disassembled after entering a normal cellular environment, reducing the probability of COSBA release. Therefore, we infer the chemotherapy dormancy characteristic of COS-BA/Ce6 NPs to the tumor microenvironment is valid and reliable, and the design idea in the present study is feasible. 

3.5. In vitro chemotherapy/PDT anticancer efficacy of COS-BA/ Ce6 NPs

Additionally, prior to detailed in vitro chemotherapy/PDT anticancer efficacy of COS-BA/Ce6 NPs and to further uncover the endocytosis mechanism, we study the four main endocytosis pathways by adding corresponding endocytic inhibitors including chlorpromazine (clathrin-mediated), chloroquine phosphate (lysosome acidification inhibitor), nystatin (caveolae-mediated), and a macropinocytosis inhibitor EIPA (ethylisopropyl amiloride) (Fig. 5C and D). We observe that the introduction of nystatin and chlorpromazine could more efficiently inhibit phagocytic efficiency than EIPA and chloroquine phosphate, showing significantly decreased cellular fluorescence compared with normal 37  C. These results indicated that COS-BA/Ce6 NPs were internalized by multiple mechanisms, mainly through the clathrin and caveolae-mediated endocytosis pathways. Since the increased 1 O2 generation via intermolecular interactions, COS-BA/Ce6 NPs exhibit significantly enhanced intercellular ROS yield than that of Ce6 as indicated by the stronger green fluorescence of the DCFH probe (Fig. 5E), implying the higher photodynamic efficiency. MTT assays demonstrate that COS-BA/Ce6 NPs under irradiation perform a remarkably enhanced inhibition effect on 4T1 cells than that of equivalent Ce6, while with relatively moderate cytotoxicity without irradiation (Fig. 5F). Significantly, the IC50 of Ce6 (0.53 mg/mL) in COS-BA/Ce6 NPs was much lower than free Ce6 (1.39 mg/mL) (Fig. 5G), suggesting remarkably improved PDT efficacy, which was further perfectly demonstrated by the cytotoxicity of COSBA/Ce6 NPs against human MCF-7 and HepG2 cancer cells (Supporting Information Fig. S19). Likewise, by comparison with free Ce6, the relatively lower IC25, IC50, and IC75 values were also observed in co-assemblies (Fig. 5G). Meanwhile, compared with Ce6 PDT alone and single chemotherapy of NPs in the dark, COS-BA/Ce6 NPs under irradiation achieved more effective combined chemotherapy/PDT against the above three cancer cells, showing a synergism (combination index: CI ˂ 1) (Fig. 5I). Besides, the live/dead cell fluorescence dual staining using calcein-acetoxymethyl ester (calcein-AM) and propidium iodide (PI) further confirmed that COS-BA/Ce6 NPs could efficiently induce cell apoptosis and necrosis, as the treated cells were almost stained with red PI fluorescence signal when cultured for an additional 10 h after irradiation (Fig. 5H). Meanwhile, nonirradiated COS-BA/Ce6 NPs (higher dose Ce6: 3.2 mg/mL) also had certain chemotherapeutic activity as seen by the AM and PI signals simultaneously. This remarkably improved PDT activity of COS-BA/Ce6 NPs was further validated by the AM/PI staining after treatment for an additional 20 h (Supporting Information Fig. S20). Moreover, when adding sodium azide (SA) and D-mannitol (DM), a separately special quencher of 1 O2 and $OH48, to the COS-BA/Ce6 NPs-PDT-treated 4T1 cells, the obviously increased cell viability implied that Type I ($OH) and Type II (1 O2) photoreactions occurred simultaneously in COSBA/Ce6 NPs-mediated PDT process (Fig. 5J). Furthermore, COS-BA/Ce6 NPs particularly possess better blood compatibility as indicated by the negligible red blood cell hemolysis (Fig. 5K), suggesting the potential for biomedical applications via intravenous administration. 

3.6. In vivo biodistribution of COS-BA/Ce6 NPs

Subsequently, prior to the in vivo distribution investigation, we next assessed the pharmacokinetics of COS-BA/Ce6 NPs by monitoring the Ce6 concentration in the blood after injection intravenously with equivalent Ce6 or COS-BA/Ce6 NPs. The Ce6 concentration-time profiles in the blood (Fig. 6A) and the comparative pharmacokinetic parameters (Supporting Information Table S1) clearly support that COS-BA/Ce6 NPs have a prolonged circulation in the bloodstream, which may allow for greater accumulation at tumor sites. In particular, COS-BA/Ce6 NPs showed quick blood elimination within 2 h, implying favorable biodegradability. Accordingly, we then investigated the in vivo biodistribution and tumor accumulation ability of COS-BA/Ce6 NPs (Fig. 6B and C). Similar to universally reported Ce6 nanocomposites33, COS-BA/Ce6 NPs can be distributed rapidly throughout the whole body. Especially during the first 4 h, assemblies exhibited obviously stronger fluorescence emission at tumor sites than free Ce6, suggesting the improved retention and tumor-targeted accumulation ability of COS-BA/Ce6 NPs, probably attributed to the passive enhanced permeability retention (EPR) effect afforded by the nanoparticles14. After 6 h postinjection, the fluorescence intensity at tumor sites gradually decreased rapidly. Notably, unlike the commonly reported nanocomposites with significantly enhanced tumor accumulation compared to free positive drug29, COS-BA/Ce6 NPs did not show very prominent accumulation at tumor sits at 24 h post-injection than free Ce6 (Fig. 6C), which was further supported by the imaging of the excised tumor (Fig. 6D and E). The excellent biodegradability of NPs may be responsible for the introduction of natural small organic molecules. This was also observed with other terpenoids small molecule-mediated nano-assemblies49. Besides, COS-BA/Ce6 NPs also showed a similar tissue distribution as free Ce6 (Fig. 6E), and the ex vivo average fluorescence intensity was generally lower than that presented in vivo, further implying good biodegradability. Additionally, considering the close fluorescence level difference between 2 and 4 h after injection of NPs and Ce6, as well as experimental operability allowing sufficient intervals for irradiation, 4 h after administration was chosen as the light time point for subsequent PDT therapy.

Considering the not very prominent tumor accumulation at 24 h than free Ce6, to further validate the increased EPR effect that COS-BA/Ce6 NPs do have, we immediately monitored the ex vivo fluorescence images of tumors at different times after injection intravenously with nanodrugs (Fig. 6F and G). Remarkably, either at 20 min or at 12 h post-injection, COS-BA/Ce6 NPs indeed showed stronger fluorescence emission at tumor sites than free Ce6, demonstrating excellent and enhanced tumor accumulation ability. Meanwhile, as time passed, the fluorescence intensities retained in tumor tissue gradually weakened (Fig. 6G), which is entirely consistent with the results of in vivo animal fluorescence imaging. Clearly, this rapid biological metabolism and enhanced tumor accumulation avoided potential long-term accumulation toxicity, guaranteeing highly efficient antitumor therapy with better biosafety.

Figure 5 In vitro anticancer efficacy of COS-BA/Ce6 NPs. Cellular internalization of (A) 4T1 cancer cells and (B) LO2 normal cells incubated in free Ce6 or COS-BA/Ce6 NPs for 0.5 and 3 h, and corresponding flow cytometric analysis of fluorescence intensity. (C) Uptake inhibition of specific endocytic pathway studies and (D) corresponding fluorescence intensity of cells measured by flow cytometry after incubation with COSBA/Ce6 NPs with/without endocytic inhibitors including chlorpromazine, chloroquine, nystatin, and ethylisopropyl amiloride (EIPA). Scale bars Z 20 mm. (E) Cellular ROS generation induced by free Ce6 or COS-BA/Ce6 NPs (equivalent Ce6: 1.6 mg/mL). Scale bars Z 20 mm. (F) Cell viability of 4T1 cells after various treatments for 24 h. (G) The IC25, IC50, IC75 analysis of free Ce6 and COS-BA/Ce6 NPs against 4T1, MCF-7, and HepG2 cells, respectively. (H) Calcein-AM/propidium iodide (PI) live/dead staining of COS-BA/Ce6 NPs (Ce6: 1.6 mg/mL)-treated 4T1 cells after incubation for additional 10 h, as well as non-irradiated COS-BA/Ce6 NPs (Ce6: 3.2 mg/mL). Scale bars Z 50 mm. (I) Combination index (CI) of COS-BA/Ce6 NPs on 4T1, MCF-7, and HepG2 cells, as determined by Chou Talalay theorem calculation. (J) PDT mechanisms (Type I and Type II) induced by irradiation of COS-BA/Ce6 NPs and evaluated by specific 1 O2 and $OH quencher sodium azide (SA) and D-mannitol (DM), respectively. (K) Percent hemolysis of red blood cells after incubation with COS-BA/Ce6 NPs for 4 h. Data are expressed as means  SD (n Z 3). *P ˂ 0.05, **P ˂ 0.01, and ***P ˂ 0.001 show the significant differences.

Figure 6 In vivo blood circulation and biodistribution of COS-BA/Ce6 NPs. (A) Ce6 concentration-time profiles in the blood after i.v. injections of equivalent Ce6 or co-assembled COS-BA/Ce6 NPs (n Z 3). (B) Whole-body fluorescence images of 4T1 tumor-bearing mice after i.v. injections of indicated drugs at various time points. (C) Average fluorescence intensity obtained from the tumor sites in (B). (D) Ex vivo fluorescence images of tumors and major organs excised from the above mice at 24 h, and corresponding (E) fluorescence intensity quantitative result (n Z 3). (F) Ex vivo fluorescence images of tumors and major organs at different times after i.v. injections of Ce6 or COS-BA/Ce6 NPs, and (G) corresponding fluorescence intensity analysis of tumors. Data are expressed as means  SD (n Z 3).

3.7. In vivo chemotherapy/PDT combinatorial anti-tumor treatment 

Motivated by the unique dormancy characteristic, good biodegradability, and PDT activity, the in vivo chemotherapy/PDT combination antitumor therapy was investigated. 4T1 tumor bearing mice were randomly divided into 6 groups and injected intravenously with equivalent Ce6 (3.5 mg/kg of body) every 2 days for a total of three doses. 4 h post-injection, the tumor sites of mice in the light groups were irradiated for 15 min (Fig. 7A). Meanwhile, corresponding tumor volumes or body weights were recorded every other day. After 14 days of treatment (Fig. 7B), free Ce6 with irradiation and free COS-BA were ineffective in tumor suppression, likely due to the limited drug accumulation in tumor tissue. In contrast, after irradiation of COS-BA/Ce6 NPs, the most significant tumor inhibition was observed, validating the excellent chemotherapy/PDT combination efficiency. The average tumor weights (Fig. 7D) and corresponding photographs of dissected tumors (Fig. 7E) both demonstrated the best antitumor efficacy of COS-BA/Ce6 NPs under irradiation, as treated mice showed the smallest tumor sizes than those of other groups. The mean tumor inhibition ratio of PDT-treated COS-BA/Ce6 NPs reached 80.3%, higher than those of free Ce6 PDT (42.4%), and free COS-BA (38.5%). Notably, although previous in vitro studies have shown that COS-BA/Ce6 NPs without light showed a dormant function of chemotherapeutic activity, they still perform an enhanced antitumor efficiency (49.5%) compared to free COS-BA (Fig. 7D). We speculated that two possible reasons contribute to this result, one is the higher tumor accumulation of COS-BA/Ce6 NPs due to the widely reported EPR effect. Another factor is the weakly acidic tumor microenvironment that stimulated the release of highly toxic COS-BA. Because of the higher critical micelle concentration (CMC, 187 mg/mL) of COS-BA (Supporting Information Fig. S21), it could exist in the monomer state in the tumor environment, thus favoring the improved chemotherapy of non-irradiated COS-BA/Ce6 NPs. Besides, to further verify the superior anticancer efficacy of COS-BA/Ce6 NPs, hematoxylin and eosin (H&E) staining of tumors was performed (Fig. 7G).

Figure 7 Efficacy study with a subcutaneous 4T1 xenograft model. (A) Schematic illustration of in vivo PDT treatment. (B) Tumor growth profiles, (C) body weight changes, and (D) excised tumor weights containing tumor inhibition ratios of 4T1-bearing mice after various treatments in the indicated groups (n Z 5 for each group). (E) Images of excised tumors from representative mice after treatment. (F) Serum biochemistry analysis after treatment with COS-BA/Ce6 NPs for 14 days. (G) Representative H&E staining of excised tumor sections after different treatments. Scale bars Z 50 mm. (H) TUNEL staining of free Ce6 and COS-BA/Ce6 NPs PDT-treated tumor sections. Scale bars Z 20 mm **P < 0.01 indicates the significant differences.

The results indicated that COS-BA/Ce6 NPs-PDT-treated tumor sections showed serious tissue deterioration and nuclei deficiency, while moderate tissue necrosis was observed in the other groups. Simultaneously, compared with free Ce6 light, COS-BA/Ce6 NPs under irradiation remarkably increased the number of TUNEL-positive cells showing a stronger brown nuclear staining (Fig. 7H), further confirming the excellent chemotherapy/PDT combination therapy. Moreover, no obvious variations of body weights were observed in all groups (Fig. 7C). Even if the COSBA/Ce6 NPs or Ce6 light groups had a modest loss in body weight during administration, the body weights returned to normal after a few days. No evident damage or inflammatory lesion could be found in the major organs harvested at the end of treatments (Supporting Information Fig. S22), both indicating the low/no systemic toxicity and good biosafety of COS-BA/Ce6 NPs. Furthermore, although COS-BA/Ce6 NPs are mainly accumulated in liver tissue, the corresponding liver function markers (ALT, AST, and ALP) did not show significant changes compared to normal healthy mice (Fig. 7F), suggesting better biocompatibility. In addition, the renal function markers (BUN, CRE, and UA) and biochemical parameters (ALB, and TBIL) also appeared to be similar to normal mice, further implying better biosafety. Clearly, this prodrug organic small molecule COS-BA-mediated co-assemblies COS-BA/Ce6 NPs possess multiple favorable therapeutic features that open up attractive possibilities for applications in clinical medicine. 

3.8. Antitumor immunity of combined chemotherapy/PDT and anti-PD-L1 blockade

Recently, numerous innovative studies have shown that the combination of cancer immunotherapy with other treatment modalities could effectively rouse tumor-specific immune responses to the elimination of primary or distant tumors1. Encouraged by the unique chemotherapy dormancy function and excellent chemotherapy/PDT antitumor efficacy of COS-BA/Ce6 NPs, we thus wonder whether it could also trigger immunological responses with the assistance of an immune adjuvant anti-PD-L1 to cause highly efficient antitumor therapy. Could it treat distant tumors that cannot be treated directly using PDT treatment, thereby providing an alternative strategy for clinical anticancer applications? To test this hypothesis, in this study, a "two tumors" model was established and detailed experiment procedures were shown in Fig. 8A. Typically, two 4T1 tumors were inoculated in both the left and right flanks of each mouse. Considering that anti-PD-L1 alone does not have a conspicuous immunotherapeutic effect50, the experiments were divided into 5 groups including an untreated group, COS-BA/Ce6 NPs, COS-BA/Ce6 NPs with anti-PD-L1, COS-BA/Ce6 NPs with light, COS-BA/Ce6 NPs with light and anti-PD-L1. After a single i.v. injection of NPs (Ce6: 3.5 mg/kg), the right tumors (primary tumor) in the light groups were irradiated for 15 min to evaluate the immunological effects of PDT/ chemotherapy, while the left tumors (distant tumors) in all groups without any treatment were selected for individual chemotherapy. Subsequently, on Days 2, 4, and 6, mice in the immune groups were i.v. injected with anti-PD-L1 antibody (15 mg per mouse per injection) for a total of three doses. Significantly, regardless of PDT treatment or not, COS-BA/Ce6 NPs plus anti-PD-L1 could efficiently inhibit the primary tumor progression (Fig. 8B). And corresponding average tumor inhibition ratios separately reached 86.7% (irradiation) and 76.5% (no irradiation), far higher than individual chemotherapy (5.7%) or chemotherapy/PDT (27.4%) treatment (Fig. 8C). The photographs of dissected tumors (Fig. 8D) and 4T1-bearing mice (Supporting Information Fig. S23) after 14 days of treatment also visually supported the above conclusion. These results suggested that both NPs-based chemotherapy and chemotherapy/PDT could effectively activate antitumor immunity. Meanwhile, most strikingly, anti-PD-L1 blockade plus NPs also offered a remarkable tumor inhibition effect to non-irradiated distant (left) tumors (Fig. 8E and F), generally stronger than COS-BA/Ce6 NPs alone, which was also confirmed by the corresponding dissected tumors and mice images after treatment (Fig. 8D and Fig. S23), further demonstrating the efficient antitumor immune response induced by COS-BA/Ce6 NPs.

To fully explore this efficient antitumor immune response, CD8þ cytotoxic T lymphocytes (CTLs), a critical immunocyte for cancer immunotherapy4, were further evaluated by analyzing the immune cells in the right or left tumors after 14 days of treatment. It was found that COS-BA/Ce6 NPs plus anti-PD-L1 with or without PDT treatment induced robust CD8þ CTL infiltration in the primary tumors (Fig. 8H and J). The CD8þ CTL infiltration was up to 11.3% after COS-BA/Ce6 NPs-based chemotherapy/ PDT plus anti-PD-L1 treatment, slightly higher than COS-BA/Ce6 NPs plus anti-PD-L1 (9.63%), and much higher than that in other groups without anti-PD-L1, including treatment with COS-BA/ Ce6 NPs-based chemotherapy (5.12%) or chemotherapy/PDT alone (4.07%). Similarly, compared to non-immune groups, significantly increased CTL infiltration was also observed in the left (distant) tumors for mice after COS-BA/Ce6 NPs plus antiPD-L1 with or without PDT treatment (Fig. 8I and K), further implying highly effective immune responses induced by chemotherapy or chemotherapy/PDT combination therapy. Meanwhile, after the introduction of anti-PD-L1, both COS-BA/Ce6-based chemotherapy and chemotherapy/PDT led to robust production of multiple cytokines that play pivotal roles in regulating the cytotoxic functions of CTLs8, including tumor necrosis factor-alpha (TNF-a) (Fig. 8L) and interferon-gamma (IFN-g) (Fig. 8M) in the serum samples of mice after 14 days of treatment, further supporting that there were efficient antitumor immune responses. Besides, no apparent changes in body weights were observed in different treatment groups (Fig. 8N), indicating the favorable biosafety of this combined chemotherapy/PDT/immunotherapy. Taken together, we found that although anti-PD-L1 plus COSBA/Ce6 NPs with light showed a slightly higher tumor inhibition ratio (86.7%) than the non-irradiated group (76.5%) against the right tumors owing to the influence of PDT (Fig. 8B‒D), no significant differences in therapeutic effects on the left tumors was observed in the presence (77.8%) or absence (78.4%) of PDT (Fig. 8E‒G). We speculate the following possible reasons lead to this phenomenon. Firstly, for the mechanism of chemotherapy or PDT enhancing the immunological effects of anti-PD-L1 checkpoint blockade, accumulating studies have demonstrated that these treatment modalities could trigger acute inflammation in the tumor area, thereby increasing the presentation of tumor-associated antigens to T cells, thus inducing cancer cells to undergo ICD8,9. Tumor cells undergoing ICD would up-regulate "eat me" signals by exposing the calreticulin (CRT) on the surfaces of immunogenically dying tumor cells and "danger" signals by releasing high-mobility group box 1 (HMGB1), promoting activation of dendritic cells to activate CTLs, whose activity could be enhanced by anti-PD-L1, thus resulting in significantly improved anticancer immunotherapy51.

Figure 8 Anti-tumor effect of COS-BA/Ce6 NPs-based chemotherapy/PDT plus checkpoint blockade anti-PD-L1 immunotherapy. (A) Schematic illustration of CNPs-based chemotherapy/PDT with anti-PD-L1 therapy. (BeD) (B) The tumor growth profiles, (C) average tumor weights, and (D) images of excised tumors from representative mice for primary (right) tumors after various treatments for 14 days as indicated. (EeG) (E) The tumor growth curves, (F) average tumor weights, and (G) images of excised tumors for non-irradiated distant (left) tumors after different treatments as indicated. (H) Populations of CD8þ T cells in right tumors and (J) percentages of CTL infiltration were quantitatively detected by flow cytometry after 14 days of treatment. (I, K) Populations of CD8þ T cells in left tumors on Day 14 were quantitatively detected by flow cytometry (n Z 3). Secretion of cytokines in sera after 14 days of treatment including (L) IFN-g and (M) TNF-a measured by ELISA assay (n Z 3). (N) Changes in mice body weight during various treatments. *P < 0.05, **P < 0.01, and ***P < 0.001 show the significant differences.

Secondly, in this work, COS-BA/Ce6 NPs could be distributed on the left and right tumors simultaneously, both the left chemotherapy and the right chemotherapy/PDT have huge potential to induce tumor cells undergoing ICD. Meanwhile, COS-BA/Ce6 NPs are administered in only one dose, the immunotherapy mainly depends on three doses of anti-PD-L1, whether chemotherapy or chemotherapy/PDT, they are only primers. The therapeutic differences in inducing tumor cell ICD between individual chemotherapy and combined chemotherapy/PDT treatments might not be very prominent. The percentage of CD8þ CTLs on the left (Fig. 8I and K) and right tumors (Fig. 8H and J) also supported the above speculation, no significant difference in CD8þ CTLs was observed in the presence or absence of PDT. Therefore, COS-BA/Ce6 NPs plus anti-PD-L1 with or without irradiation achieved similar anticancer efficiency against the left tumors. Furthermore, because 4T1 cells express low levels of PD-L152, anti-PD-L1 alone has been widely proven to have any conspicuous immunotherapeutic effect on 4T1 breast cancers53,54. Despite the ICD effects mediated by COS-BA/Ce6 NPs warrant further investigation, the presented results have shown that COS-BA/Ce6 NPs achieve remarkably enhanced immune responses with the assistance of anti-PD-L1. Collectively, it seems that COS-BA/Ce6 NPs indeed potentiated the cancer immunotherapy of anti-PD-L1, not just a combination therapy. Although anti-PD-L1 plus COS-BA/Ce6 NPs with or without PDT treatment did not show significant differences in therapeutic effects, both of them showed obviously enhanced antitumor efficacy compared to one dose of COS-BA/Ce6 NPs. That is to say, even if without light treatment, once the immune adjuvant antiPD-L1 was introduced, the immunogenicity of tumor residues after COS-BA/Ce6 NPs chemotherapy can be boosted resulting in remarkable systemic antitumor immune responses, thus leading to effective suppression of primary or distant tumors. Thus, it should be possible to eliminate primary and metastatic tumors during clinical anticancer immunotherapy with repeated treatments with NPs. Clearly, these results collectively provide evidence of highly effective and synergistic antitumor immune responses induced by COS-BA/Ce6 NPs in combination with an anti-PD-L1 blockade, with either chemotherapy alone or in combination with chemotherapy/PDT.

cistanche tubulosa-improve immune system

4. Conclusions

In summary, we purposefully designed, synthesized, and developed a biocompatible, biodegradable, low toxic but highly efficient and clinically available transformed photo-chemotherapeutic nano-prodrug COS-BA/Ce6 NPs by combining three multifunctional components self-assembled natural small molecule betulinic acid (BA), a water-soluble COS, and a low toxic photosensitizer Ce6 for combined chemotherapy/PDT. It subsequently triggers highly effective and synergistic antitumor immune responses in combination with an anti-PD-L1 blockade, with either chemotherapy alone or in combination with chemotherapy/PDT. Specifically, we show that hollow network molecular arrangement might facilitate the formation of shell-structured nanoparticles. Modifications of water-soluble COS could greatly enhance the anticancer activity of lipophilic natural small molecules by constructing an amphiphilic prodrug. We also show that the resulting nanodrugs showed multiple favorable therapeutic features, especially a smart "dormancy" function with insidious chemotherapy effects. Combined with the distinctive pH responsiveness, these co-assemblies perform low toxicity and biosafety with normal tissue but are highly toxic to tumor tissues. Together with the improved 1 O2 generation, good biodegradability, and biocompatibility, all of which contributed to the highly efficient, synergistic, and secure anticancer chemotherapy/PDT/ immunotherapy. In particular, individual chemotherapy without PDT treatment can activate systemic antitumor immune responses, opening up attractive possibilities for clinically synergistic anticancer chemotherapy immunotherapy. Overall, this work provides promising insight for developing natural multifunctional highly bioactive, and low-toxic nano-immunostimulants for clinical immunotherapy.

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