A targeting nanoplatform for chemo-photothermal synergistic therapy of small-cell lung cancer

doi:10.21203/rs.3.rs-3106535/v1

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A targeting nanoplatform for chemo-photothermal synergistic therapy of small-cell lung cancer

https://doi.org/10.21203/rs.3.rs-3106535/v1

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published 10 Jul, 2024

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The chemo-photothermal cotherapy has created a breakthrough in cancer treatment, but the precise delivery of drugs to tumor sites and the thermoresistance of tumors remain major challenges in photothermal therapy (PTT). Due to its overexpression in small cell lung cancer (SCLC) and limited normal tissue expression, Somatostatin receptor 2 (SSTR2) is proposed as an ideal target for the precise treatment of SCLC. We first developed a novel anti-SSTR2 monoclonal antibody (MAb), which showed strong and specific affinity toward SSTR2 receptor overexpressed on SCLC cells. Then, a targeting nano-drug delivery system comprising anti-SSTR2 MAb surface-modified poly (lactic-co-glycolic acid) (PLGA) nanoparticles co-encapsulating Cypate and gambogic acid (GA) is prepared for achieving specific tumor targeting and chemo-photothermal combination therapy. The formed SGCPNs nanoparticles demonstrated excellent monodispersity, physiological stability, preferable biocompatibility, and resultant efficient photothermal conversion efficacy. Confocal microscopy images showed that SGCPNs were quickly internalized by SSTR2-overexpressing SCLC cells, triggering the release of GA under acidic and near-infrared (NIR) laser irradiation environments, leading to their escape from lysosomes to the cytosol and then diffusion into the nucleus. At the same time, GA molecules can not only decrease the cell survival rate but also inhibit the activity of HSP90 to enhance PTT efficacy. SGCPNs can be precisely delivered to xenograft tumors of SSTR2-positive SCLC in vivo and decrease the undesirable effects on healthy organs. Upon NIR laser irradiation, therapy of SGCPNs showed significant tumor regression. In conclusion, SGCPNs provide a new chemo-photothermal synergistic treatment strategy for targeting SCLC.

Somatostatin receptor 2

chemo-photothermal combination therapy

heat shock protein 90

gambogic acid

small cell lung cancer

Small cell lung cancer (SCLC) is the most lethal lung cancer, characterized by rapid expansion and widespread metastasis of cells with high grade poorly differentiated neuroendocrine features[1]. Radiotherapy, chemotherapy, and surgery are traditional treatments for SCLC. However, most cases with the development of metastases are inoperable, so systemic chemotherapy is always given priority to SCLC treatment. Moreover, patients could experience high toxicity and intolerance, with very limited clinical benefits during treatment, resulting in a 5-year survival rate below 10%[2]. Clearly, continuous development of new therapeutic strategies is required to enhance clinical outcomes and provide precise therapy against SCLC.

Up to now, the synergistic approach of combinational chemo-photothermal therapy has received increasing attention due to its improved antitumor therapy effect[3]. Photothermal therapy (PTT) is an emergent strategy that utilizes photothermal agents to convert photon energy into heat under near-infrared (NIR) light for tumor ablation. It offers advantages such as non-invasiveness, high therapeutic efficiency, and high security[4, 5]. Although using nanoparticles as PTT agents is feasible for the treatment of lung cancer[6], their treatment performance is still unsatisfactory due to inadequate tumor accumulation, low photothermal conversion efficiency, and inherent thermal resistance of tumors. When cancer cells are thermally stimulated, heat shock proteins (HSPs) are significantly up-regulated and thereby participating in thermal-induced autoinhibition and enhancing against heat stress of tumor cells[7]. Among different HSPs, heat shock protein 90 (HSP90) has been found as the primary contributor involved in the repair of heat damage[8]. Based on this rationale, reducing or inhibiting overexpressed HSP90 may effectively raise the heat sensitivity of tumors and thereby improving the treatment efficacy of PTT[9]. However, it is hard to ablate tumor cells completely by PTT alone[10], while the combination of PTT and chemotherapy can afford synergetic therapeutic efficacy[11]. The research found that gambogic acid (GA), a natural anti-tumor chemotherapeutic agent, holds significant anti-neoplastic activity against several types of malignant tumors and gets excellent anticancer treatment results[12, 13]. More importantly, GA can also inhibit HSP90 and then restore the cell sensitivity to heat, thus enhancing the PTT effect[14]. The attractive characteristics of GA may represent one promising strategy for alleviating the heat resistance function of tumor cells and achieving enhanced PTT efficiency. Nonetheless, the poor water solubility, lack of tumor targeting, and severely high toxicity of GA limited its clinical adoption[15]. To overcome these issues, the nanocarrier-based antitumor therapeutic strategy that overcomes its limitations and improve bioavailability has been considered promising[16].

The design of proper multifunctional nanosystems is always in high demand in the field of nanomedicine because they must be efficiently delivered to the target lesion and accumulate there to reach the treatment concentration. Nanoparticles passively accumulate at tumor sites via the enhanced permeability and retention (EPR) effect, which is due to extensive angiogenesis that enhanced permeability of vessels in tumor tissues and poor lymphatic clearance leads to the localization of the nanoparticles, decreasing side effects on healthy tissues[17]. Nevertheless, nanoparticles relying on a passive targeting mechanism usually faced with low accumulation efficiency at tumor sites, thus severely undermining their therapeutic efficiency[18]. Nowadays, active targeting strategies are widely used to enhance therapeutic effects i.e. a particular ligand conjugation on the surface of nanoparticles in order to recognize a specific receptor aberrantly expressed on tumor cells. Somatostatin receptor 2 (SSTR2) belongs to G-protein–coupled receptors family and it is overexpressed in various tumors (e.g., pancreatic tumors and neuroendocrine tumors) with limited expression in normal tissue[19]. In addition, many studies have shown that SSTR2 is highly expressed in high-grade pulmonary neuroendocrine, including SCLC[20], suggesting that it is the potential target for the development of therapeutic agents to treat SCLC. Clinicians have been using a somatostatins analog linked to 68Gallium to identify patients with SSTR2 expressing in SCLC via a routinely available imaging diagnostic technique[21], that is readily available to screen patients who may benefit from targeted therapy to treat SSTR2. In addition to diagnosis and detection, the miniature drug conjugate SSTR2-targeted PEN-221, which incorporates the SSTR2 agonist [Tyr3, Cys8] octreotate and the microtubule agent DM1, has been developed to treat SCLC[22], further establishing the value of SSTR2 as a viable target for the treatment of SCLC. A myriad of targeting ligands, such as peptides, glycoproteins, and carbohydrates, monoclonal antibodies (MAb), are the proper ligands due to their high specificity and affinity[23]. Although antibody-drug conjugates (ADCs) comprised of anti-SSTR2 MAb and monomethyl auristatin E have been developed to efficiently target neuroendocrine tumors[24], anti-SSTR2 MAb modified nanosystems for SCLC treatment has not yet been reported to best of our knowledge.

Built on the above concerns, we have exploited the electric fusion scheme to develop a new anti-SSTR2 MAb, which can be used as a specific targeting ligand towards SSTR2 for the diagnosis and therapy of SCLC due to its high affinity with NCI-H524 cells (high SSTR2 receptor expression). Cypate, a carboxyl derivative of indocyanine green used as a photothermal agent, which could be encapsulated in the micelles to trigger the PTT of tumor cells under NIR light irradiation[25]. In this study, we designed and fabricated Cypate and GA-loaded anti-SSTR2 MAb-modified poly (lactic-co-glycolic acid) (PLGA) nanoparticles (denoted as SGCPNs) for more selectively delivering therapeutic agents to the SCLC cells and achieving on-demand drug release under intracellular lysosomal acidity and a NIR laser. As indicated in Scheme 1, PLGA constituted the shell structure of hybrid nanoparticles, which could be applied to encapsulate Cypate and GA. Anti-SSTR2 MAb is modified on the surface of nanoparticles to achieve receptor-mediated targeting effects. In the targeting nanoparticles, GA serves as an HSP90 inhibitor and an antitumor drug, and Cypate acts as a photothermal agent for the photothermal effect. After tail intravenous injection, the resultant polymeric nanoparticles could accumulate at the tumor site through tumor homing capacity of SSTR2, apart from the EPR effect, and distribute into the endosomal/lysosomal via the endocytosis mechanism. SGCPNs can smoothly convert the absorbed optical energy into heat for triggering the release of GA under NIR laser irradiation, leading to lysosome escape. The released GA could not only exhibit a significant chemotherapeutic effect but also enhance PTT efficacy by inhibiting the overexpressed HSP90, thereby getting synergistic anti-tumor effects. The in vitro and in vivo antitumor therapy of SGCPNs render distinct advantages, confirming the efficient targeting ability and excellent HSPs inhibition facilitated the PTT effect. In general, our easily made SGCPNs were able to provide a unique strategy enabling targeted SCLC and chemo-photothermal combination therapy in the clinic.

Scheme 1. Schematic illustration of SGCPNs the preparation process and the function for NCI-H524 tumor cells.

2.1 Materials

Poly (lactic-co-glycolic acid)-COOH (PLGA-COOH, lactide: glycolide = 50:50, MW = 34,000) was purchased from Daigang Bio-Engineer Co., Ltd. (Jinan, China). gambogic acid (GA) and Cypate were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). The anti-HSP90 antibody, anti-GAPDH antibody, and HRP-labeled goat anti-mouse IgG secondary antibody were purchased from Abcam Corporation. Polyvinyl alcohol (PVA) and cell counting kit-8 (CCK-8) were purchased from Sigma-Aldrich. All chemicals and organic solvents used were of analytical grade.

2.2 Generation of anti-SSTR2 MAb

Anti-SSTR2 MAb was previously prepared in our laboratory. We obtained mouse hybridoma cell lines with the best anti-SSTR2 antibody expression using optimizing electric fusion parameters. The MAb was determined by enzyme-linked immunosorbent assay (ELISA) and identified by flow cytometry, which had specific binding to human SCLC cell lines (NCI-H524) cells but low binding to human normal lung epithelial cells (Beas2B). It was purified from a hybridoma culture medium using two steps by ammonium sulfate precipitation with affinity chromatography on the protein A column (manuscript in preparation).

2.3 Preparation of GCPNs

GCPNs were fabricated through an oil-in-water (O/W) emulsion technique. Briefly, PLGA-COOH (20 mg) was dissolved in 4 mL of dichloromethane as the oil phase, after which 2 mg of Cypate and 1 mg of GA were added to the organic phase. The mixture solution was quickly transferred into 10 mL of an aqueous 4% PVA water solution and homogenized for 10 min at 9500 rpm to produce the O/W emulsion, which was then dripped into a 2% isopropyl alcohol solution and stirred for 6 h at room temperature to remove dichloromethane. The GCPNs were obtained through centrifugation for 10 min at 15000 g force, washed three times using double distilled water and dispersed in water then stored at 4°C. Blank PLGA nanoparticles (denoted as BPNs), the Cypate loaded PLGA nanoparticles (denoted as CPNs), and GA loaded PLGA nanoparticles (denoted as GPNs) or Cy3.5 loaded BPNs(denoted as Cy3.5PNs)were also fabricated in the same manner.

2.4 Anti-SSTR2 MAb conjugation to GCPNs

The surface of GCPNs was functionalized with anti-SSTR2 MAb for bioconjugation employing the carbodiimide chemistry method[26]. First, 10 mg of GCPNs were centrifugated at 15000 g for 10 min and resuspended in 15 mL of MES buffer (0.1 M, pH = 5.5), and then NHS and EDC (with the molar ratio PLGA: EDC: NHS in 1:10:4) added to the nanoparticle dispersion under slow stirring for 30 min. The activated nanoparticles were obtained through centrifugation at 15000 g for 10 min and washed thrice in double deionized water to remove the excess of EDC and NHS, and then dispersed in 15 mL MES buffer (0.1 M, pH = 8.0). Next, 1mg anti-SSTR2 MAb was mixed with the solution of activated nanoparticles and then kept at 4°C for 12 h. Finally, the unbound antibodies were separated from GCPNs-SSTR2 (denoted as SGCPNs) solution by centrifugation. The purified pellet of SGCPNs was preserved at 4°C until further use. The fabrication of functionalized BPNs nanoparticles (SBPNs) and functionalized Cy3.5PNs nanoparticles (SCy3.5PNs) was the same as the above method.

2.5 Physical-chemical characterization

2.5.1 Particle size, zeta potential, morphology analysis, and stability

The morphological features of the nanoparticles were observed through scanning electron microscopy (SEM), transmission electron microscopy (TEM), and an optical microscope. The average size, polydispersity index (PDI), and zeta potential of all formulated nanoparticles diluted in deionized water were determined by dynamic light scattering (DLS). To evaluate the stability of SGCPNs, the mean diameters of the as-prepared nanoparticles in water at 4 ℃, and in PBS solution containing 10% FBS at 37℃ after storage for different periods were measured by DLS.

2.5.2 Evaluation of the encapsulation efficiency (EE) and drug loading (DL)

The supernatants of the emulsion (including the wash process) were collected during the preparation of GCPNs and SGCPNs. The absorption spectra of GA, Cypate, GCPNs. and SGCPNs were recorded using a UV − visible spectrometer (UV-3500, Agilent, USA) at 360 or 785 nm. The DL % and EE % were calculated using the following formulae:

DL % = (Weight of encapsulated drug) / (Weight of feeding drug and polymers) × 100%

EE % = (Weight of encapsulated drug) / (Weight of feeding drug) × 100%

2.5.3 Evaluation of MAb on the surface of nanoparticles

The covalent conjugation of anti-SSTR2 MAb to nanoparticles was indirectly proven by fluorescence microscopy. The Cy3.5-labelled goat anti-mouse IgG was incubated with BPNs and SBPNs for 1 h, separately. After centrifugation and washing, the sediment was collected and observed using a fluorescence microscope. The binding efficiency of anti-SSTR2 MAb to nanoparticles was further confirmed by flow cytometry. Anti-SSTR2 MAb activity on the surface of nanoparticles was analyzed using indirect ELISA. The protein amount of SBPNs was determined by a micro BCA Protein Quantification Kit at 562 nm.

2.6 In vitro photothermal effect

The different concentrations of SGCPNs (0、2.5、5、10、20 µg/mL) were placed in quartz cuvettes and then irradiated with an 808 nm NIR laser at 1.2 W/cm² for 10 min, and the temperature change was recorded at an interval of 1 min. Next, SGCPNs (Cypate concentration = 10 µg/mL) was irradiated with different output powers (0.4, 0.8, 1.2, 1.6 W/cm²), and the temperatures were also measured using the same method. Furthermore, the photothermal stability of free Cypate and SGCPNs was evaluated by detecting the photothermal state of four cyclic heating. The thermographic maps and maximum temperature were recorded using an infrared (IR) thermal imaging camera (PTi120, Fluke, USA).

2.7 In vitro drug release

The in vitro release study of GA and Cypate from SGCPNs was determined using a dialysis method. SGCPNs solution placed into dialysis bags (cut-off 10 kDa) were immersed into 50 mL of pH 7.4 or 5.5 PBS solutions, subsequently kept shaking at 37 ℃, respectively. To further investigate drug release under laser irradiation, the samples were irradiated at a power density of 1.2 W/cm² for 10 min every 30 min. Then, 5 mL of the release buffer was withdrawn at the predetermined time points and replaced with the same volume of fresh buffer. Finally, the released amount of GA and Cypate was determined by UV-Vis spectroscopy. Each sample was performed in triplicate.

2.8 Western blot assay

The NCI-H524 and Beas2B cells were purchased from the American Type Culture Collection (ATCC). The cells were cultured in RPMI-1640 media comprising 8% fetal bovine serum (FBS) at 37 ℃ with 5% CO₂.

The expression levels of SSTR2 in NCI-H524 and Beas2B cells were confirmed by western blot. First, cells were washed with PBS, then lysed with lysis buffer containing phosphatase and protease inhibitors. The protein concentration of NCI-H524 and Beas2B cells were determined by BCA Protein Quantification Kit. 10–20 µg protein of each sample was separated by SDS-PAGE experiment. The protein samples were transferred onto polyvinylidene fluoride membranes, which were blocked using 5% BSA dissolved in PBST solution. Subsequently, the membranes were incubated with anti-SSTR2 MAb at 4 ℃ for 12 h, followed by the secondary antibody at room temperature for 2 h. The membranes were processed with ECL reagent and imaged by the BIO-RAD ChemiDoc Touch imaging system. To investigate the influence of GA on the expression level of HSP90, the HSP90 protein of NCI-H524 cells and the tumor tissues were extracted after different treatments. HSP90 expression was examined using the corresponding primary antibodies, and other conditions were similar to those mentioned above. GAPDH was set as the loading control in all experiments.

2.9 Cellular uptake and internalization of nanoparticles

The cellular uptake behaviors of SGCPNs and GCPNs in Beas2B and NCI-H524 cells were studied using flow cytometry and confocal laser scanning microscopy (CLSM). The fluorescent probe Cy3.5 was encapsulated into the SBPNs and BPNs instead of GA and Cypate as previously described, while the obtained nanoparticles were abbreviated as SCy3.5PNs and Cy3.5PNs. Beas2B and NCI-H524 cells at a density of 5×10⁴ cells/mL were seeded into 24-well plates containing CLSM culture dishes in each well. The next day, the cells were incubated with SCy3.5PNs or Cy3.5PNs at a Cy3.5 concentration of 500 ng/mL in the media for 2 h. The inhibition group of cells was incubated with excessive anti-SSTR2 MAb (1 mg/mL) for 2 h, followed by the addition of the SCy3.5PNs for an additional 2 h. After the incubation, the cells were rinsed with PBS and fixed with 500 µL of 4% paraformaldehyde for 30 min. DAPI was used to stain the cell nuclei in the dark for 5 min and fluorescence images were observed using a CLSM (Olympus Corporation, Japan). DAPI and Cy3.5 showed blue and red colorations, respectively. As mentioned above, the cells were digested with trypsinase and incubated with SCy3.5PNs and Cy3.5PNs for 1 h and 2 h, respectively. Thereafter, the fluorescence intensity of Cy3.5 in the cells was analyzed using the flow cytometer (BD Accuri C6, USA).

For evaluating the intracellular distribution of nanoparticles after NIR laser irradiation, SGCPNs were added to CLSM dishes containing NCI-H524 cells. The cells were incubated with SGCPNs at 37 ℃ for 4 h and subjected with or without the NIR laser irradiation, and then stained with Lysotracker Green (100 nM) for 0.5 h. The cells were stained with DAPI and then observed under a CLSM.

2.10 Cytotoxicity and apoptosis study

NCI-H524 and Beas2B cells were seeded in 96-well plates (5×10³ cells/well) and incubated under standard conditions (37 ℃ and 5% CO₂) until they attached. For evaluating the biocompatibility of the samples, the various concentrations of BPNs, CPNs, and SBPNs were added into NCI-H524 and Beas2B cells for 24 h incubation. For evaluating the cytotoxicity of GA, the various concentrations of SGCPNs, GCPNs, GPNs, and free GA were added into NCI-H524 cells at 37 ℃ for 24 h incubation. In addition, to demonstrate combination therapy in vitro, the various concentrations of free GA, CPNs, and SGCPNs were added into another group of cells at 37 ℃ for 4 h incubation. Cells were irradiated with 808 nm laser for 10min at 1.2 W/cm² or left untreated, and cultured for another 24 h. The relative cell viability was investigated by a CCK-8 assay. The absorbance value (OD_450nm) was determined for each well by a multifunctional microplate reader (Molecular Devices, USA). The viability (%) of cells was calculated by the following formula:

Cell viability (%)=(OD_{450nm(sample)}-OD_450nm(blank))/(OD_{450nm(control)}-OD_450nm(blank))×100, where OD_{450nm(sample)}, OD_{450nm(control)}, and OD_450nm(blank) refer to the absorbance of the experimental group, the control group, and the blank wells, respectively. To achieve the visualization of cytotoxicity, NCI-H524 cells were treated using the above mentioned method, and then stained for 30 min with CAM and PI for further observation using CLSM.

For verifying the apoptosis efficiency of cells, NCI-H524 cells seeded in 6-well plates were incubated with different nanoparticles or exposed to NIR laser irradiation (808 nm, 1.2 W/cm², 10min). Cell apoptosis was analyzed with the following eight treatments: PBS, Laser, GPNs, GCPNs, SGCPNs, CPNs + Laser, GCPNs + Laser, and SGCPNs + Laser. Afterward, a total of 1×10⁶ cells were collected in each group and washed with PBS. Then, Annexin V-FITC and PI were added following the procedure of the manufacturer before detected using flow cytometry.

2.11 Intratumoral distribution of nanoparticles

Female BALB/c nude mice (18 ± 3g) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. All experimental protocols utilizing animals were performed based on the Care and Use of Laboratory Animals, approved by the Animal Experimentation Ethics Committee of Jilin Medical University in China. To establish the tumor model, approximately 5×10⁶ NCI-H524 cells were subcutaneously injected into the flank of nude mice. When the tumor grew to a size of approximately 100 mm³, tumor-bearing mice were administrated with SGCPNs and GCPNs (Cypate: 2 mg/kg) through the tail vein. The mice were anesthetized, and imaged at pre-scheduled time intervals using an IVIS Lumina LT3 Imaging System (Perkin Elmer, USA). After the last imaging, the mice were sacrificed, and the main organs (heart, liver, spleen, lung, and kidney) as well as the tumor, were removed and visualized using the same system as described above. To further analyze the targeting and penetration of SGCPNs and GCPNs into tumor tissues, tumors were paraffin-embedded and sectioned. The nuclei were stained with DAPI, and the distribution of nanoparticles was visualized using CLSM.

2.12 In vivo chemo-photothermal combination therapy

Mice bearing NCI-H524 tumors were randomized into six groups, and injected via tail vein with PBS, Laser, SGCPNs, GCPNs + Laser, GPNs, and SGCPNs + Laser. The various formulations of nanoparticles (Cypate: 2 mg/kg, GA: 1 mg/kg) were injected every three days for a total of four treatments, respectively, and irradiated with a 10 min of 808 nm irradiance of 1.2 W/cm². The temperature change was evaluated using an infrared (IR) thermal imaging camera every 2 min. Subsequently, mice weight and tumor volume were determined every 2 days after different treatments. The tumor volume (V) was evaluated by the following expression: V= (L×W²)/2, where W and L represent the shortest and the longest tumor diameters, respectively. All the mice were euthanized on 2 days after the last dose, and tumor tissues and major organs were gathered for hematoxylin and eosin (H&E) stain. Moreover, the proliferating cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were carried out on tumor slices for the evaluation of cell proliferation and apoptosis. Blood samples of treated mice in each group were assessed for biochemical and hematological examination.

2.13 Statistical analyses

All experimental data were presented as mean ± SD. Statistical significance was tested by GraphPad Prism 5.01 version. Statistical analysis was analyzed using the student's t-test for the differences between various treatments, and values of p < 0.05 indicated statistically significant.

3.1 Preparation and characterization of nanoparticles

Considering that hydrophobicity and nonspecific toxicity of Cypate and GA limited clinical translation in cancer treatment, the water-soluble GCPNs were prepared using the single-emulsion method, and anti-SSTR2 MAb was conjugated to the surface of GCPNs via an EDC/NHS reaction. The morphology of nanoparticles (Fig. 1A), as observed by optical microscopy, exhibited a spherical shape and high dispersity. As shown in TEM images (Fig. 1B-C and Figure S1), the GCPNs, SGCPNs, and BPNs had a uniform dispersion without noticeable adhesion and a well-defined spherical morphology. As shown in the TEM images of SGCPNs (Fig. 1D), the inner black dense core was speculated to encapsulate GA or Cypate, and the outer shell of the gray-black portion could contain anti-SSTR2 MAb. The mean particle size, surface charge, and polydispersity index (PDI) of nanoparticles, assessed by DLS, are presented in Table 1. The DLS analysis indicated that the mean diameters of the BPNs, GCPNs, and SGCPNs were 113.7 ± 9.7, 156.6 ± 5.2 and 217.3 ± 8.4 with zeta potential of -5.6 ± 0.2, -9.8 ± 0.6 and − 11.5 ± 0.5, respectively. The decrease in the surface charge and increase in size for the functionalized particles indirectly revealed that successful loading of GA and Cypate and grafting of anti-SSTR2 MAb. Nanomedicine particles with sizes ranging from 10 to 500 nm can preferentially access the tumor tissue via the leaky tumor vasculature[27], and the active targeting achieved by SGCPNs might somewhat enhance drug accumulation at tumor regions[28]. The negative zeta potential is conducive to ensuring the uniform dispersion of nanoparticles and thereby preventing aggregation in the suspension[29]. In addition, SGCPNs maintained a relatively stable nanoscale size after storage at 4 ℃ for 14 days (Fig. 1E). More importantly, SGCPNs were stored in the PBS solution containing 10% FBS for 24 h at 37 ℃, the particle size remained unchanged, confirming that SGCPNs had excellent stability (Figure S2).

As illustrated in Fig. 1F, the solution of BPNs appeared as a white emulsion, and the GPNs showed a homogeneous light-yellow emulsion, while the color of the GCPNs suspension turned from light-yellow to dark green after loading Cypate. The loading of Cypate/GA was verified using the UV-vis absorbance of SGCPNs where the absorption peaks of SGCPNs was at 360 nm and 801nm, but the absorption peak characteristic of Cypate shifted from 780 to 801nm (Fig. 1F). The red-shifted of maximum absorption peak is likely attributed to the influence of the solvent effect or the intermolecular interplay[30, 31]. The DL and EE of GA in SGCPNs were measured to be 3.33 ± 0.28% and 70.08 ± 1.26%, respectively, while those for Cypate were 6.61 ± 0.17% and 72.68 ± 2.52% (Table 1). The high EE values of the polymeric nanoparticles validated that SGCPNs could be used as delivery carriers with a significant amount of drug GA/Cypate encapsulation.

Table 1

Characterization of CGPNs, SCGPNs and BPNs nanoparticles
Nanoparticles	Size(nm)	PDI	Zeta Potential(mV)	GA		Cypate
Nanoparticles	Size(nm)	PDI	Zeta Potential(mV)	EE(%)	DL(%)	EE(%)	DL(%)
BPNs	113.7 ± 9.7	0.25 ± 0.03	-5.6 ± 0.2	-	-	-	-
CGPNs	156.6 ± 5.2	0.32 ± 0.07	-9.8 ± 0.6	82.96 ± 3.12	3.95 ± 0.57	81.65 ± 4.33	7.42 ± 0.35
SCGPNs	217.3 ± 8.4	0.43 ± 0.04	-11.5 ± 0.5	70.08 ± 1.26	3.33 ± 0.28	72.68 ± 2.52	6.61 ± 0.17
Data are the mean of three determinations ± SD

3.2 Verification of MAb conjugation to the surface of nanoparticles

The Cy3.5-labeled secondary antibody was used to confirm the conjugation of anti-SSTR2 MAb to the nanoparticles′ surface. As depicted in (Fig. 2A-B), unlike BPNs, the SBPNs revealed intense red fluorescence, which resulted from the secondary antibody absorbed directly on the surface of nanoparticles by binding specifically to anti-SSTR2 MAb. In addition, the flow cytometry in Fig. 2C-D showed that the fluorescence intensities of the SBPNs and BPNs were approximately 86.79% and 2.58%, respectively, further indicating that practically all nanoparticles are anti-SSTR2 MAb coated. The results shown in Fig. 2E, the ELISA assay indicated that SBPNs specifically bind to SSTR2 protein, confirming the functionalization, while the same amount of anti-SSTR2 MAb was included as a positive control. Furthermore, the concentration of anti-SSTR2 MAb on the surface of nanoparticles was about 0.52 mg/mL through the BCA assay (Fig. 2F).

3.3 The photothermal properties and thermal sensitivity of nanoparticles

To examine the photothermal conversion properties of SGCPNs at different Cypate loading concentrations, they were irradiated with an 808 nm irradiance of 1.2 W/cm². As shown in Fig. 3A, the solution temperature of SGCPNs at Cypate concentrations of 20 µg/mL increased significantly from 26.3 to 51.6 ℃ after 10 min of irradiation, whereas negligible temperature changes were observed for deionized water under the same irradiation condition, which proved that the SGCPNs solution exhibited a typical concentration-dependent manner. Meanwhile, the photothermal properties of SGCPNs would increase correspondingly with increasing laser power density (Fig. 3B). The infrared thermal images in Fig. 3C showed that there was evident heat absorption in SGCPNs solution with an increase in irradiation time, indicating that the photothermal properties of SGCPNs were superior to free Cypate. The superior photothermal conversion capacity of SGCPNs should be likely attributed to the improved stability of Cypate that was encapsulated within polymeric nanoparticles, with a higher condensed concentration than free Cypate, which results in SGCPNs having more heat radiation and less heat dissipation after laser irradiation[32–34]. To verify the light stability of SGCPNs, the absorption value of Cypate was first detected upon 808 nm laser irradiation (1.2 W/cm²). The absorption value of free Cypate significantly decreased after 6 min of NIR irradiation due to photobleaching defects of Cypate upon the light irradiation (Figure S3A). However, the SGCPNs could effectively increase the photostability of Cypate (Figure S3B). Next, SGCPNs showed well-repeated temperature elevation compared to the free Cypate under the consecutive laser for four on/off cycles, which more intuitively reflected their excellent photostability (Fig. 3D). All these results revealed that SGCPNs have outstanding photothermal properties and may be implemented as a robust heat generator for PTT.

3.4 In vitro light-triggered drug release study

Considering that lysosomal compartments were slightly acidic in tumor cells[35], the GA release behavior from SGCPNs was determined in different PBS solutions under simulated physiological conditions. Figure 4A depicts that GA release rates from SGCPNs were pH-dependent. The initial accelerated release of GA started after 24h, and a sustained release reached up to 32.37% at pH 7.4 and 56.83% at pH 5.5 after 72 h, respectively. The initial release has been believed to be the release of drug adsorbed onto the particles′ surface[36]. The sustained release of GA can be ascribed to the hydrolytic degradation of the PLGA and the drug diffusion from the polymer[36]. Hydrolysis of PLGA was increased at a lower pH value, thus, the release rate of GA was accelerated when exposed to acidic pH, which was consistent with previous reports[36]. Next, the drug release effect was studied by irradiating SGCPNs at pH 5.5 without or with NIR laser irradiation. As indicated in Fig. 4B, the GA release of SGCPNs was significantly accelerated during the laser-on period as compared to the laser-off period, which implied that laser irradiation could further facilitate drug release. The spherical structure of SGCPNs collapsed, and the particle size tended to increase and became irregular after NIR light irradiation under an acid environment (Figure S4), further suggesting that the acidic conditions and NIR laser irradiation could result in disruption of the nanocarrier structure and fast release of the drug. We speculated that SGCPNs nanoparticles exhibited excellent stability and prolonged release of the drug under normal conditions, while reducing the drug release of GA in blood and normal tissues.

3.5 Cellular uptake, intracellular localization, and endosomal escape ability of nanoparticles

The NCI-H524 cell has a high SSTR2 expression level on the cellular membrane, while Beas2B, a human bronchial epithelial cell line, is SSTR2 negative[37]. In our study, NCI-H524 and Beas2B were selected as model cells, and SSTR2 expression was firstly evaluated through western blot. As presented in Figure S5, SSTR2 expression was higher in NCI-H524 cells than in Beas2B cells, indicating that anti-SSTR2 MAb can be applied as targeted ligands for tumor-specific intracellular delivery of anticancer drugs.

To validate the targeted effect of anti-SSTR2 MAb, the interactions between nanoparticles and cell lines (NCI-H524 and Beas2B) were evaluated by flow cytometry and CLSM, using Cy3.5-loaded nanoparticles. From CLSM images (Fig. 5A), stronger red fluorescence signals around the nucleus, as well as the cytoplasm of NCI-H524 cells were obtained in the SCy3.5PNs group, whereas a weak fluorescent signal around the cell surfaces was obtained in the Cy3.5PNs group. In the inhibition group, SCy3.5PNs lost the ability to target NCI-H524 cells since SSTR2 proteins were blocked by excess-free anti-SSTR2 MAb, while the fluorescence signals were approximately the same as observed for the nontargeted group. This indicated that SCy3.5PNs could enhance targeting efficiency by receptor-mediated active targeting for the SSTR2 overexpressed lung cancer cells. Comparatively, for Beas2B cells with low SSTR2 expression, both SCy3.5PNs and the Cy3.5PNs exhibited poor fluorescence signals.

Flow cytometry further indicated that SCy3.5PNs displayed a strong fluorescence intensity of 47.1% and 98.1%, while Cy3.5PNs displayed a fluorescence intensity of 11.3% and 21.9% in 1h and 2 h, respectively, in NCI-H524 cells (Fig. 5B). The cellular uptake of SCy3.5PNs was approximately 4-fold greater than that of the nontargeted Cy3.5CPNs, suggesting that SCy3.5PNs efficiently accumulated in the NCI-H524 cells. It's worth noting that SCy3.5PNs exhibited uptake efficiency, which was similar to that of Cy3.5PNs in Beas2B cells. The existing literature reported that PLGA nanoparticles are taken up by vascular smooth muscle cells and cancer cells through energy-dependent endocytic pathways[38, 39]. However, receptor-mediated endocytosis enhanced the internalization rate and specificity of nanoparticles compared with adsorptive endocytosis[40]. The high internalization rate of anti-SSTR2 MAb mediated endocytosis explains the higher uptake of SCy3.5PNs compared to Cy3.5PNs into NCI-H524 cells.

As displayed in Fig. 5C, significant yellow fluorescence signals from the overlap of the green (representing lysosome) and red (SGCPNs) fluorescence signals were generated with a Pearson's correlation coefficient (PC) of 0.48 without NIR laser irradiation, indicating the selective localization of SGCPNs in the lysosome[41, 42]. On the contrary, no overlapping signals (PC = 0.15) were generated in NCI-H524 cells under NIR laser irradiation, clearly showing that separate the red dots were spread extensively outward rather and moved toward the nucleus than being distributed in the Lysotracker Green regions. The disruption of lysosomes could translocate more nanoparticles under NIR laser irradiation, while much of the drug was released from the nanoparticles and could easily escape from lysosomes to the cytoplasm, which primarily caused cytotoxic effects[34]. Based on these facts, we speculated that the presence of anti-SSTR2 MAb on the surface of nanoparticles favored enhanced cellular targeting uptake efficiency and further selectively localized in the lysosomes of NCI-H524 cells. SGCPNs displayed the burst release under acidic conditions and NIR laser irradiation. The high amount of GA could escape from lysosomes to the cytoplasm, and diffuse into the nucleus, potentially facilitating the therapeutic efficacy of synergistic therapy.

3.6 In vitro mechanisms of self-sensitized PTT and synergistic photothermal cytotoxicity of nanoparticles

Encouraged by the above results, the therapeutic efficacy of various formulations of nanoparticles was analyzed against NCI-H524/Beas2B cells by the CCK-8 assay. This assay cannot only evaluate the biosecurity assay but also identify differences among them. First, various samples of BPNs, CPNs, and SBPNs without NIR laser irradiation were non-cytotoxic to NCI-H524 cells at different concentrations (Figure S6A), as well as in normal cells Beas2B, as expected (Figure S6B). This indicated that PLGA-based nanoparticles were less toxic and possessed excellent biocompatibility for application in vivo and that CPNs was also nontoxic to the cells without NIR irradiation. Moreover, the targeted nature of SBPNs did not exhibit a concentration-dependent increase in cytotoxicity between cell lines, regardless of noticeable differences in SSTR2 expression, indicating that the presence of anti-SSTR2 MAb did not promote toxicity of nanoparticles.

Next, to determine the viabilities of tumor cells after treatment with SGCPNs, GCPNs, GPNs, and GA, NCI-H524 cells were incubated with different concentrations of nanoparticles. As seen in Fig. 6A, these formulations revealed concentration-dependent cytotoxicity of GA, indicating the prominent anti-tumor capability of GA. No significant change occurred in the cell viability for untreated cells. By contrast, the relative viabilities of cells incubated with free GA and SGCPNs significantly decreased under the same condition, while SGCPNs treated cells exhibited a higher decrease in viability compared to free GA, which was also due to antibody-mediated endocytosis enhanced internalization of SGCPNs. These differences were not noticeable between GCPNs and GPNs, which was ascribed to the incompletion release of GA from nanoparticles. When cancer cells are thermally stimulated by stressful conditions, they increase HSPs expression, such as HSP90, to mitigate the injury incurred by stress[43]. GA, as a potential inhibitor of HSPs, could disturb the normal functions of HSPs and reduce the thermoresistance of tumor cells[44]. Thus, western blot was further used to characterize the expression of HSP90 in NCI-H524 cells after various treatments of control, GA group, CPNs + Laser group, and SGCPNs + Laser group. The free GA group exhibited significantly decreased intracellular HSP90 levels compared to all other groups, and the amount of HSP90 produced after NIR laser irradiation in the SGCPNs group were slightly less than in the control group (Fig. 6B and C). The expression of HSP90 only increased greatly in the CPNs group with laser, further proving that GA could inhibit HSP90 expression. Considering the strong absorption of Cypate in the NIR region, we next studied whether GA-induced down-regulation of HSP90 expression could be utilized to increase the PTT efficiency of SGCPNs. It was found that SGCPNs + Laser group treated cells showed a remarkable decreased viability in comparison to cells incubated with free GA, SGCPNs, and CPNs + Laser group. These results suggested that the fast released of GA molecules from SGCPNs under NIR laser irradiation further blocked HSP90 expression and suppressed the thermoresistance of tumor cells, making it possible to enhance the PTT effect (Fig. 6D). Notably, the cell viability of the SGCPNs + Laser group was dramatically decreased, which also further delineated that chemo-photothermal combination therapy by SSTR2 targeting contributes to improving the anti-tumor therapeutic efficacy. In addition, the cell toxicity of different formulations in Beas2B cells was also evaluated. Just as shown in Figure S6C, different treatments showed little toxicity to Beas2B cells, demonstrating that nanoparticles cannot cause damage to healthy tissues.

Furthermore, to visualize the synergistic therapeutic efficacy of chemo-photothermal combination therapy, NCI-H524 cells were stained with CAM and PI to identify live cells (green fluorescence) and dead cells (red fluorescence), which were observed by CLSM (Figure S7). From these results, dead cells were barely observed in the PBS and NIR laser irradiation group, part of the NCI-H524 cells in the CPNs + Laser group, GPNs group and SGCPNs group emitted red fluorescence, which indicated that a few cells have been killed. Notably, massive cells emitted red fluorescence in the SGCPNs + Laser group, indicating a higher cell death rate. To confirm the above results, the apoptosis of NCI-H524 cells was studied using the Annexin V-FITC/PI test. As demonstrated in Figs. 6E and 6F, the control group and the group subjected only to NIR laser irradiation exhibited no significant apoptosis ratio. The percentages of apoptotic cells (early and late) were 27.8% for GCPNs, while the cell apoptosis percentage of GCPNs + Laser increased to 39.1%. Moreover, compared to other treatment groups, SGCPNs + Laser showed the highest total apoptotic ratio of 48.2%. These results all verified that combination multimodal therapy presented more advanced tumor treatments.

3.7 In vivo imaging and tissue distribution of nanoparticles

Excellent tumor accumulation is expected to enhance the efficiency of chemo-photothermal combination therapy. Based on the results of fluorescence imaging (Figs. 7A and 7B), bright fluorescence signals were observed in the liver at 3 h after administration. Over time, compared to the small amount of red fluorescence signals of the GCPNs group, a large amount of fluorescence signal of the SGCPNs group was generated after 6 h intravenous administration and continued to increase without a significant reduction in the tumor region until 24 h, revealing that grafting of the SSTR2 antibody substantially improved tumor-targeting abilities of nanoparticles. To more precisely monitor the targeting capability in vivo, tumors and the major organs (heart, liver, spleen, lung, kidney) were taken out for ex vivo imaging at 24 h post-injection. SGCPNs were basically accumulated in livers, lungs, and tumors and then produced a stronger fluorescence signal in the tumor, while GCPNs caused less tumor accumulation (Fig. 7C). Nanoparticles were accumulated in both the liver and lung because those tissues are the typical reticuloendothelial system[45]. GCPNs passively targeted the tumor tissues through the EPR effect[46], whereas SGCPNs were actively targeted by anti-SSTR2 MAb surface modification. Besides, the semi-quantitative analysis of the fluorescent signal in Fig. 7D revealed that the intratumoral average fluorescence intensity of SGCPNs was approximately 1.6-fold greater than that of GCPNs, further confirming the excellent in vivo tumor targeting of SGCPNs. The target and penetration ability of SGCPNs in vivo were also evaluated on sections of the tumor using CLSM. As depicted in Fig. 7E, considerable amounts of red fluorescence representing SGCPNs were found around tumor cells in the targeted group, which was more than that in the non-targeted group. Therefore, these results demonstrated that SGCPNs have excellent targeting ability and significant anti-tumor efficiency can be easily realized.

3.8 In vivo the combined antitumor therapy efficacy, thermal imaging, and safety evaluation

Considering the most direct validation approach for assessing chemo-photothermal combination therapy of nanoparticles is in vivo photothermal efficacy[47], the antitumor efficacy was studied in NCI-H524 tumor-bearing nude mice. Thermographic images (Fig. 8A) and real-time temperature (Fig. 8B) of the tumor site were monitored during NIR laser irradiation. SGCPNs showed a higher temperature at the tumor site(>50 ℃)than that of GCPNs (around 43 ℃), which was sufficient to induce irreversible damage in tumor tissues[48], indicating that the SGCPNs not only could efficiently target the tumor regions in vivo but also achieved the excellent tumor ablation. In contrast, the tumor temperature showed little change in mice receiving PBS treatment under the same laser irradiation conditions.

After administering different treatments, tumor sizes were monitored by vernier calipers throughout 14 d, and photographs of typical mice in different groups were recorded to show the treatment effect (Fig. 8C and Figure S8A). The tumors of the laser-only group grew rapidly, and the PBS group showed a similar trend, which confirms that NIR laser irradiation without Cypate had scarcely any impact on tumor growth. GPNs and SGCPNs showed a limited therapeutic outcome as there was no photothermal effect to increase tumor efficacy. Moreover, all the mice in the GCPNs + Laser group began to recur at 8 d and the tumor growth rate increased, illustrating that the insufficient accumulation of nanoparticles induced tumor recurrence because of the incomplete ablation. As expected, the mice treated with SGCPNs under NIR laser irradiation exhibited more effective tumor growth inhibition due to targeting and synergistic chemo-photothermal, indicating that SGCPNs take advantage of SSTR2-mediated active targeting, allowing high tumor-specific accumulation with sustained GA release in situ as well as HSP90 inhibition to augment PTT. In addition, digital images and tumor weights of excised tumors from sacrificed mice were recorded (Figure S8B and 8D), obtaining a similar tendency with tumor volume assay. The synergistic antitumor effects of SGCPNs were further assessed by histologic and immunohistochemical examination, as displayed in Fig. 8E. H&E staining revealed that the most severe tumor cell damage (changed cell shapes, nuclei lysis, and chromatin condensation) was found in the SGCPNs + Laser group, whereas cancer cells in the GCPNs + Laser, GPNs, and SGCPNs group were only partially destroyed, and no notably effects were observed in the other 2 groups. TUNEL apoptosis staining showed the same trend as H&E staining, with the maximal number of apoptotic cells of SGCPNs + Laser group in the tumor sections. The results of PCNA staining also revealed the potent tumor-inhibiting capacity of SGCPNs under NIR laser irradiation, with the fewest tumor cell proliferation. We subsequently detected HSP90 expression levels in tumors from all groups using the western blot assay. The tumor tissues treated with GCPNs + Laser had higher levels of HSP90 expression, demonstrating that the up-regulation of HSP90 in the tumor cells could be triggered by the PTT-induced heating stress in vivo (Figure S9A, B). Notably, treatment with GPNs and SGCPNs significantly decreased the HSP90 expression in tumor cells, further confirming that the GA introduction could markedly suppress the activity of HSP90 in vivo. These results provided a piece of direct evidence for combination multimodal therapy based on HSP90 inhibition.

Next, changes in body weight were detected to evaluate the biosafety of SGCPNs during the treatment. There were no obvious changes in the mice body weights in all treatment groups (Fig. 8F), verifying no/low systemic toxicity of the nanoparticles in vivo. To further evaluate the off-target toxicity and biocompatibility of the nanoparticles, the main organs of mice were dissected for H&E staining. As displayed in Figure S10, there was no obvious damage or inflammatory reaction in the main organs after the various treatments, suggesting no obvious toxicity and good biocompatibility of SGCPNs. We further investigated the potential cytotoxicity of the different treatments through hem analysis and biochemical analysis. As shown in Figure S11A, there were no remarkable differences in related biochemical indices among all mice, demonstrating the undamaged functions of the liver and kidney. These indicators showed no significant differences and all were within the normal range among treatment groups, verifying good hemocompatibility of nanoparticles (Figure S11B). These results strongly supported the fact that the excellent biocompatibility of SGCPNs ensures the feasibility of in vivo applications, guaranteeing their further clinical translation.

In summary, SGCPNs is the multifunctional nanoparticles with a shell spherical structure, targeting property, and synergistic therapy with HSPs inhibitor by PTT. This study has the following highlights and conceptual advances: (i) anti-SSTR2 MAb offering targetability of functionalized nanoparticles; (ii) synergistic PTT of HSPs inhibitor GA; (iii) High therapeutic biosafety. Briefly, SGCPNs with biodegradable had a shell for easy surface modification, successfully loaded the hydrophobic GA and Cypate, linked with anti-SSTR2 MAb by connecting the carboxyl groups and the amino, and allowed long-time circulation. Despite previous studies have reported that HSPs inhibition have been adopted to enhance the PTT effect[49], the NIR penetration capacity was usually challenged by depth-attenuation of light, which limited clinical application potential[50]. In contrast, due to the excellent targeting and chemo-photothermal synergistic therapy ability of SGCPNs, a sufficient number of nanoparticles reach deep-seated tumors to achieve the optimal temperature and overcome the depth-attenuation effect of light. SGCPNs can be efficiently taken into tumor cells through SSTR2 targeting and be accumulated in the lysosome while showing remarkable hyperthermia and lysosomes escape upon NIR laser irradiation. In addition, SGCPNs can also release GA triggered by NIR laser irradiation within tumor tissues to kill the remaining alive tumor cells after laser irradiation and inhibit tumor recurrence. Collectively, the strategy lays the foundation for achieving satisfactory chemo-photothermal synergistic therapeutic efficacy owing to the rational application of the material characteristics, promising future clinical translation.

Overall, the anti-SSTR2 MAb modified active targeting nano-platform (SGCPNs) could efficiently co-encapsulate the heat shock protein inhibitor GA and the photothermal agent Cypate to realize targeting and chemo-phototherapy synergistic therapy. Obviously, SGCPNs exhibited good stability, biocompatibility, photothermal effect, and pH-dependent and laser-triggered drug release. The surface of particles was modified with anti-SSTR2 MAb to target tumor cells, which remarkably improved their accumulation in SSTR2 overexpressing cells. The introduced GA exerted a significant chemotherapeutic effect while blocking the stressfully overexpressed HSP90 to reverse the thermoresistance of tumor cells, thereby enhancing the PTT therapeutic efficacy. In vivo experiments demonstrated that the targeting ability of SGCPNs overcame the limitation of nanoparticles effectively accumulating in the tumor regions while reducing the distribution of GA in healthy organs. More critically, SGCPNs presented remarkable inhibition of tumor growth based on combined targeting and chemo-photothermal therapy. Accordingly, the current study provided a multifunctional nanosystem with a targeting capability to enhance the therapeutic effect of NIR photosensitizers through the decrease of HSP90, which showed massive potential for clinically precise therapy of SCLC.

Authors’ contributions

Guangyao Kong acquired funds and managed the project. Moli Yin, Lei Liu, Wenliang Li and Yu Yan designed, executed, wrote and revised manuscripts. Huiyan Wang and Yuan Dong provided revision guidance and part of the financial support. All authors discussed the results and approved final version.

Conflicts of interest

The authors declare that they have no conflict of interest.

Acknowledgments

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Key Research and Development Program of China (grant no. 2019YFA0801804). Jilin Provincial Department of education (grant nos. JJKH20230535KJ and JJKH20210489KJ). Research and Development of Industrial Technology Program of Jilin Province (grant no. YDZJ202102CXJD048). Jilin province science and technology project of traditional Chinese medicine (grant no. 2022087). National Training Program of Innovation and Entrepreneurship for Undergraduates (grant no. S202213706034).

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Scheme 1 is available in Supplementary Files section.

No competing interests reported.

Scheme1.tif
Scheme 1. Schematic illustration of SGCPNs the preparation process and the function for NCI-H524 tumor cells.
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A targeting nanoplatform for chemo-photothermal synergistic therapy of small-cell lung cancer

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Abstract

Figures

1. Introduction

2. Materials and method

2.1 Materials

2.2 Generation of anti-SSTR2 MAb

2.3 Preparation of GCPNs

2.4 Anti-SSTR2 MAb conjugation to GCPNs

2.5 Physical-chemical characterization

2.5.1 Particle size, zeta potential, morphology analysis, and stability

2.5.2 Evaluation of the encapsulation efficiency (EE) and drug loading (DL)

2.5.3 Evaluation of MAb on the surface of nanoparticles

2.6 In vitro photothermal effect

2.7 In vitro drug release

2.8 Western blot assay

2.9 Cellular uptake and internalization of nanoparticles

2.10 Cytotoxicity and apoptosis study

2.11 Intratumoral distribution of nanoparticles

2.12 In vivo chemo-photothermal combination therapy

2.13 Statistical analyses

3. Results and discussion

3.1 Preparation and characterization of nanoparticles

3.2 Verification of MAb conjugation to the surface of nanoparticles

3.3 The photothermal properties and thermal sensitivity of nanoparticles

3.4 In vitro light-triggered drug release study

3.5 Cellular uptake, intracellular localization, and endosomal escape ability of nanoparticles

3.6 In vitro mechanisms of self-sensitized PTT and synergistic photothermal cytotoxicity of nanoparticles

3.7 In vivo imaging and tissue distribution of nanoparticles

3.8 In vivo the combined antitumor therapy efficacy, thermal imaging, and safety evaluation

4. Conclusion

Declarations

References

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