Porous PLGA microspheres for the inhalation delivery of icariin and miR-23b in the treatment of metastatic lung cancer

Boyu Xiong; Xinxin Shao; Guangxu Fang; Mengmeng Dong; Haobo Han; Quanshun Li

doi:10.1016/j.ajps.2024.101008

. 2024 Dec 11;20(2):101008. doi: 10.1016/j.ajps.2024.101008

Porous PLGA microspheres for the inhalation delivery of icariin and miR-23b in the treatment of metastatic lung cancer

Boyu Xiong ^a, Xinxin Shao ^a, Guangxu Fang ^a, Mengmeng Dong ^a, Haobo Han ^a,^⁎, Quanshun Li ^a,^b,^⁎

PMCID: PMC12125581 PMID: 40453754

Abstract

Herein, porous poly(lactic-co-glycolic acid) (PLGA) microspheres were prepared to load icariin and miR-23b for the treatment of metastatic lung cancer. The microspheres exhibited desirable aerodynamic diameter, high drug loading and encapsulation efficiency, as well as a favorable drug release profile, which was beneficial for the deposition and exposure of drugs in the lung tissues. The release solution from microspheres exhibited a favorable anti-proliferative effect by inducting cell apoptosis and arresting the cell cycle at G1 phase, and meanwhile inhibited the migration and invasion of cancer cells. More importantly, the microspheres could be effectively inhaled and accumulated in the lung tissues to trigger the in situ apoptosis of tumor cells and suppress metastasis, using mice bearing melanoma-metastatic lung cancer as a model. Furthermore, inhalation of the microspheres showed favorable biocompatibility, barely causing tissue damage. Overall, porous PLGA microspheres provide a promising platform for the inhalable co-delivery of drugs and genes to obtain ideal therapeutic efficacy in lung cancer and other pulmonary diseases.

Keywords: PLGA microsphere, Icariin, miR-23b, Inhalation, Metastatic lung cancer

Graphical abstract

Porous PLGA microspheres harboring icariin and PEI/miR-23b nanoparticles were prepared by double emulsification/solvent evaporation method. They could be well inhaled and deposited in the lungs after the proper aerolization, thereby obtaining favorable efficacy in the treatment of metastatic lung cancer.

1. Introduction

Lung carcinoma remains the leading cause of cancer-related death worldwide with 1.80 million deaths in 2020 [1]. Currently, patients suffering from lung cancer could be offered with one or more different treatments depending on the type and stage of disease, including chemotherapy, surgery, radiotherapy and immunotherapy [2]. Among them, chemotherapy plays a key role in the treatment of lung cancers, but the chemotherapeutics have several drawbacks hindering the continuous treatment and lowering the quality of patients’ life, such as cumulative toxicity to normal tissues and multidrug resistance [3,4]. Thus, it is of great significance to achieve the combination of chemotherapy and gene therapy, which could reduce the dose of chemotherapeutics, improve the anti-tumor efficacy, increase the mutation difficulty of tumor cells, and even reverse the multidrug resistance [5].

Icariin (ICA) is a key compound extracted from Epimedium [6], and has been identified as the major bioactive ingredient with anti-cancer effect due to the induction of apoptosis and cell cycle arrest as well as the immunomodulation [7,8]. Moreover, ICA showed obvious anti-angiogenic effect by disrupting the newly formed blood vessels in tumor tissues, thereby inhibiting the migration and invasion of tumor cells [9]. However, the anti-tumor mechanism of ICA was complex, for example, the improved expression of SIRT6 and other factors related to the endoplasmic reticulum stress [10]. The combination of ICA and gene therapy is potential to act on tumor cells through different pathways and mechanisms, thereby forming a multi-target regulatory network to enhance the tumor-killing efficacy and overcome the limitations of single strategy.

Among the therapeutic genes, microRNAs (miRNAs), a class of short endogenous RNAs of 12–22 nucleotides in length, could post-transcriptionally alleviate the gene expression by the mode of RNA interference, and their dysregulation will lead to the proliferation and metastasis of tumor cells [[11], [12], [13]]. miR-23b is usually downregulated in various tumors and confirmed as a tumor suppressor to inhibit the cell proliferation and invasion [[14], [15], [16], [17]]. Meanwhile, the overexpression of miR-23b significantly enhanced the sensitivity of tumor cells towards cisplatin, implying that miR-23b could be applied as an adjuvant enhancer to potentiate the anti-cancer efficacy [18]. In our previous report, the intracellular delivery of miR-23b into cancer cells could achieve an efficient anti-cancer response by inducing cell apoptosis and cell cycle arrest, suggesting that the miR-23b delivery is a promising strategy to execute the cancer gene therapy [19]. Although miR-23b alone could trigger anti-cancer response, the tumor cells still partially escaped from the miR-23b treatment leading to the recurrence of cancer, mainly owing to the complex regulation network of cancers and the inherent genetic redundancy of miRNAs.

Currently, the systemic administration of therapeutics is still the major approach for the treatment of lung cancers, leading to strong toxicity and insufficient drug accumulation in lung tissues [20]. Alternatively, pulmonary administration as a non-invasive approach offers benefits in the treatment of lung cancers by the direct inhalation of therapeutic agents to lung tissues, thereby obtaining high drug concentration in tumors, prolonging the drug retention and avoiding the systemic adverse effects of intravenous administration [21,22]. To date, porous poly(lactic-co-glycolic acid) (PLGA) microspheres have been widely used as potential pulmonary delivery systems in the treatment of various lung diseases such as lung cancer, asthma, tuberculosis and chronic obstructive pulmonary disease due to the characteristics of low density, ideal aerodynamic diameter, favorable lung deposition and excellent biodegradability [[23], [24], [25]]. Moreover, the negatively charged PLGA was found to possess negligible toxicity, which barely induced the inflammation in the alveolar macrophages [26]. Besides, the porous structure endowed the microspheres with more intracavity space for the encapsulation of drugs and genes, which was beneficial for rapid lung deposition and sustained release of cargoes [27,28]. To date, the delivery of doxorubicin and/or miR-519c has been successfully performed using porous PLGA microspheres or functional mixed-polymer microspheres [29,30]. Also, electrospraying porous microspheres have been prepared to load oridonin for the local therapy of primary lung cancer via pulmonary delivery [31]. However, porous PLGA microspheres have not been used in the co-delivery of traditional Chinese medicine compounds and miRNAs especially applied at an in vivo level.

Herein, inhalable PLGA microspheres harboring ICA and polyethylenimine (PEI)/miR-23b nanoparticles were prepared through double emulsification/solvent evaporation method. The porous PLGA microspheres were characterized including morphology, aerodynamic diameter, zeta potential and drug loading. The anti-cancer efficacy of microspheres was tested both in human lung adenocarcinoma A549 cells and mice bearing melanoma-metastatic lung cancer in an inhalation manner. Finally, the in vivo biodistribution and biocompatibility of microspheres were evaluated.

2. Materials and methods

2.1. Materials

PLGA (Mw = 1.1 kDa) with lactic acid/glycolic acid ratio of 50:50 was acquired from Daigang Biomaterial Co. (Jinan, China). ICA and fetal bovine serum (FBS) were obtained from MeilunBio (Dalian, China) and Kangyuan (Tianjin, China), respectively. The construction of pcDNA-miR-23b plasmid was carried out by GenePharma (Suzhou, China), which was amplified in Escherichia coli DH5α and purified by Vigorous Plasmid Maxprep kit (Beijing, China). Phosphate buffered saline (PBS) and Dulbecco's modified Eagle's medium (DMEM) were provided by Gibco (Grand Island, NY). 3-(4,5-Dimethylth-iazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) and 4,6-diamidino-2-phenylindile (DAPI) were acquired from Amersco (Solon, OH). Rhodamine B isothiocyanate (RBITC) and coumarin-6 were purchased from Rhawn (Shanghai, China) and Aladdin (Shanghai, China), respectively. The Quant-It PicoGreen dsDNA kit and LIVE/DEAD® Viability/Cytotoxicity kit and Hoechst 33,342 were purchased from Thermo-Fisher (Eugene, OR). The kits for monitoring the activities of caspase-3, −8 and −9 were acquired from Promega (Madison, WI). Branched PEI (Mw = 25 kDa) was provided by Sigma-Aldrich (Saint Louis, MO). The JC-1-based mitochondrial membrane potential assay kit and one-step terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay kit were obtained from Beyotime (Nanjing, China). Annexin V-FITC/propidium iodide (PI) apoptosis and PI-based cell cycle detection kits were purchased by Bestbio (Shanghai, China). The primary antibodies against procaspase-3, -8 and -9, Cyclin D1, Survivin, Notch-1, MMP-9, PTEN, Bcl-2, Ki67 and β-actin, and horseradish peroxidase (HRP)-labeled secondary antibodies were purchased from Abcam (Shanghai, China). The primary antibody against PARP and Cy3-labeled secondary antibody were acquired from Cell Signaling Technology (Shanghai, China) and Servicebio (Wuhan, China), respectively. Unless otherwise mentioned, all the reagents were purchased with the highest grade and used without further purification.

2.2. Preparation of porous PLGA microspheres

The porous PLGA microspheres were constructed by double emulsification/solvent evaporation method as described in our previous report [32], and the microspheres were named as follows: blank, blank porous PLGA microspheres; PPM-1, ICA-loaded porous PLGA microspheres; PPM-2, PEI/miR-23b-loaded porous PLGA microspheres; PPM-3, porous PLGA microspheres harboring ICA and PEI25K/miR-23b. In brief, 100 mg PLGA and 5 mg ICA were successively dissolved in 2 ml dichloromethane, after which 200 µl distilled water containing PEI25K/miR-23b nanoparticles (1.5:1, w/w; 40 µg plasmid) was added. The mixture was homogenized at 8,000 r/min for 30 s on ice bath, and then 100 µl ammonium bicarbonate solution (50 mg/ml for PPM-1, and 30 mg/ml for PPM-2 and PPM-3) was added into the mixture which was homogenized at 8,000 r/min for additional 30 s. The sample was injected into 50 ml poly(vinyl alcohol) (PVA) solution (1.0%, w/v) by a syringe. After the homogenization at 4,000 r/min for 1 min, the obtained emulsion was stirred at 25 °C for 3 h to remove the residual dichloromethane. Subsequently, the microspheres were collected by centrifugation at 4,000 r/min for 5 min and the supernatant was harvested to measure the drug loading and encapsulation efficiency. Finally, the microspheres were rinsed with distilled water three times and lyophilized for further use. Other porous PLGA microspheres including RBITC-PEI/miR-23b- and coumarin-6-loaded microspheres were prepared by the same procedure.

2.3. Characterization of porous PLGA microspheres

The porous PLGA microspheres were subjected to the morphologic analysis by JSM-IT500A scanning electron microscope (SEM, JEOL, Japan) at an accelerating voltage of 10 kV. The particle size was measured using the software Nano Measurer 1.2 (http://nano-measurer.software.informer.com/) by randomly selecting 1,000 microspheres from SEM images. The aerodynamic diameter (d_aero) was calculated according to the well-established formula [32].

d_{a e r o} = \frac{d}{γ} \sqrt{ρ / ρ_{a}}

In this equation, d represented the geometric diameter, γ was the dynamic shape factor (1.0 for spheres), and ρ and ρ_a (1.0 g/ml) represented the microspheres’ tap density and water mass density, respectively.

2.4. Drug loading and encapsulation efficiency measurement

The drug loading (DL) and encapsulation efficiency (EE) of ICA were calculated by the measurement of ICA concentration by reverse-phase high-performance liquid chromatography (RP-HPLC) at 30 °C on a Shimadzu SPD-20 RP-HPLC equipped with Symmetry C₁₈ column (WondaCract ODS-2, 4.6 mm × 150 mm, 100 Å). Acetonitrile/water (30:70, v/v) was used as a mobile phase at a flow rate of 1.0 ml/min, monitoring at 270 nm. The DL and EE values of miR-23b were determined based on the plasmid concentration measured by Quant-It PicoGreen dsDNA kit.

2.5. Cellular uptake of the components released from porous PLGA microspheres

To detect the endocytosis of components released from porous PLGA microspheres, coumarin-6 was incorporated to replace ICA to prepare coumarin-6-loaded porous PLGA microspheres, and RBITC was used to label PEI for the preparation of RBITC-PEI/miR-23b-loaded porous PLGA microspheres. The fluorescence of microspheres was observed by IX73P1F fluorescence microscopy (Olympus, Tokyo, Japan). First, A549 cells were seeded in 6-well plates at a density of 2.0 × 10⁵ cells/well and cultured at 37 °C for 24 h. The cells were then treated with the release solution at Day 8 from these two types of microspheres for 48 h, washed with PBS twice and stained with Hoechst 33342 at 25 °C for 30 min. The cellular uptake of coumarin-6 and RBITC-labeled PEI/miR-23b was assayed using LSM 710 confocal laser scanning microscope (CLSM, Carl Zeiss Microscopy LLC, Jena, Germany). Finally, the cells were harvested to determine the endocytosis by CytoFLEX flow cytometer (Beckman Coulter Inc., Kraemer Boulevard Brea, CA).

Total miRNAs were extracted from A549 cells by miRNA extraction kit (DP501, TIANGEN, Beijing), and the cDNA product was synthesized by miRNA First-strand cDNA Synthesis kit (B532451, Sangon, Shanghai). Afterward, qPCR was performed by SG Fast qPCR Master Mix (High Rox) (2×) on Applied Biosystems QuantStudio™ 3 (Foster City, CA). The relative expression of miR-23b was normalized by 2^−ΔΔCt method using U6 gene as the reference (primer sequence: 5′-CATCACATTGCCAGGGATTACC-3′).

2.6. Anti-proliferative analysis

The inhibition of cell proliferation induced by the released solutions from porous PLGA microspheres was evaluated by MTT assay in 96-well plates. Briefly, A549 cells with an initial density of 8.0 × 10³ cells/well were cultured overnight, and then all the wells were replenished with 200 µl DMEM containing 50 µl of release solutions obtained from porous PLGA microspheres at different days for 48 h. After the treatment with MTT solution (20 µl, 5 mg/ml) for 4 h, the solution in each well was discarded, and dimethyl sulfoxide (150 µl) was added into each well to dissolve the formazan crystals. Finally, the absorbance at 492 nm was measured by an HBS-1096A microplate reader (Nanjing, China) to calculate the cell viability, which was equal to the ratio of absorbance in the treated and untreated groups.

2.7. Cell apoptosis and cell cycle arrest analysis

Briefly, A549 cells were seeded in 6-well plates at a density of 2.0 × 10⁵ cells/well and incubated at 37 °C for 24 h. Then the cells were treated with 2 ml DMEM harboring 500 µl release solution from porous PLGA microspheres collected on Day 8. After the incubation for 48 h, the cells were collected by centrifugation at 1,500 r/min for 5 min and washed with cold PBS. For the cell apoptosis, the harvested cells were stained with FITC-conjugated Annexin V and PI solution at room temperature for 30 min. For the cell cycle arrest, the cells were fixed with ethanol at −20 °C, centrifugated at 2,000 r/min for 5 min, and incubated with RNase and PI solution at 37 °C for 30 min. Finally, the stained cells were analyzed by CytoFLEX flow cytometer (Beckman Coulter Inc., Kraemer Boulevard Brea, CA).

2.8. Western blotting analysis

The A549 cells were cultured and treated with different release supernatants from microspheres as described in the Section 2.6. The collected cells were lysed by radio immuno precipitation assay (RIPA) buffer containing 1 mM phenylmethylsulphonyl fluoride (PMSF) on ice for 1 h. Then the lysate was centrifugated at 12,000 r/min for 10 min to obtain the supernatant. The concentration of total proteins was quantified by BCA method. Afterwards, equal amount of proteins was subjected to SDS-PAGE analysis at 180 V for 150 min and transferred to PVDF membranes, which were blocked by 5 ml PBST buffer (PBS with 0.05% Tween-20) with 5% (w/v) skim milk at 25 °C for 3 h. Subsequently, the membranes were incubated with primary antibodies at 4 °C overnight, washed with PBST three times and incubated with HRP-labeled secondary antibody (1:5000 dilution) at room temperature for 1 h. Finally, the blots were washed with PBST and treated with enhanced chemiluminescence solution. The bands of specific proteins were visualized by Tanon-2500 gel image analysis system (Shanghai, China), and quantified using Image J software (v1.8.0, NIH, USA).

2.9. Wound healing assay

The inhibition of cell migration was evaluated through wound healing assay. Briefly, A549 cells were seeded in a 6-well plate at an initial density of 3.0 × 10⁵ cells/well and incubated until the confluence reached 95%. Subsequently, the cells were scratched by a sterilized 200-µl pipette tip to form uniform wounds. After removing the unattached cells by PBS, the adherent cells were treated with 2 ml fresh DMEM containing 200 µl release solution. The change of wound areas was monitored at 0, 12, 24, 36, 48 and 72 h through an IX73P1F fluorescence microscopy (Olympus, Tokyo, Japan). Three random zones were selected to calculate the average width of cell migration.

2.10. In vivo biodistribution of porous PLGA microspheres

All animal experiments were conducted according to the “Guide for the Care and Use of Laboratory Animals” (8th edition, International Publication No: 978–0–309–15,400–0) and approved by the Animal Welfare Ethics Committee of Jilin University (license No SYXK(JI)2021–0142). Female BALB/c mice (16–18 g) were supplied by Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The mice were anesthetized by isoflurane and administered with coumarin-6-loaded microspheres, RBITC-labeled PEI/miR-23b-loaded microspheres, and the microspheres harboring coumarin-6 and RBITC-labeled PEI/miR-23b via an intratracheal route by a Penn-Century DP-4 dry powder insufflator (Philadelphia, PA). At predetermined periods (0 h, 6 h, 24 h, 48 h, 5 d, 7 d and 10 d), the mice were sacrificed, and the main organs (heart, liver, spleen, lung and kidney) were excised and washed with saline. The fluorescence signals of coumarin-6 (E_x = 455 nm, E_m = 506 nm) and RBITC (E_x = 550 nm, E_m = 600 nm) were detected by IVIS® Lumina XR Spectrum system (PerkinElmer, Hopkinton, MA).

2.11. In vivo anti-tumor efficacy of porous PLGA microspheres

The in vivo anti-tumor efficacy of different porous PLGA microspheres via pulmonary administration was investigated using B16F10 melanoma lung metastasis mice. Briefly, female BALB/c mice (16–18 g) were intravenously injected with B16F10 cells (2.0 × 10⁵ cells per mouse) to establish the metastatic lung cancer model. After 7 d, tumor-bearing mice were randomly divided into 5 groups (n = 4 per group), whereas a group of mice without the implantation of B16F10 cells were served as the healthy control (n = 5). On Day 7, 14 and 21 after the implantation of cancer cells, the tumor-bearing mice were placed in a supine position and administered with the following PLGA microspheres via intratracheal route by a Penn-Century DP-4 dry powder insufflator (Philadelphia, PA): air (served as negative control), blank microspheres (5.00 mg microspheres each mouse), PPM-1 microspheres (4.80 mg microspheres containing 97.64 µg ICA each mouse), PPM-2 microspheres (4.63 mg of microspheres containing 1.48 µg miR-23b each mouse), and PPM-3 microspheres (5.00 mg of microspheres containing 1.48 µg miR-23b and 97.64 µg ICA each mouse). On Day 28, the mice were sacrificed and their lung tissues were excised. The area of metastatic nodules and the weight of lung tissues were measured to assess the therapeutic efficacy of porous PLGA microspheres. Additionally, the lung tissues were fixed with 4% (v/v) paraformaldehyde (PFA) and embedded into paraffin. The embedded tissues were then sectioned into 4-µm slides for haematoxylin and eosin (H&E) staining and observed by IX73P1F fluorescence microscopy (Olympus, Tokyo, Japan). Similarly, another batch of animal experiment was conducted to evaluate the in vivo anti-tumor efficacy of porous PLGA microspheres via pulmonary administration. Briefly, the inoculation number of B16F10 was 5.0 × 10⁵ cells for each mouse (n = 5 per group), and the intervention time was extended to five weeks.

2.12. Immunohistochemical (IHC) analysis of metastatic melanoma

The sections of lung tissues were deparaffinized, rehydrated and heated in EDTA antigen retrieval buffer (pH 8.0) for 8 min. The endogenous peroxidase of tissues was blocked with 3% hydrogen peroxide for 10 min. Subsequently, the sections were incubated with 10% goat serum for 30 min to block the nonspecific binding of antibody. To evaluate the cell proliferation, the sections were incubated with Ki-67 primary antibody at 4 °C overnight and washed with PBS three times. Then the slides were incubated with the secondary antibody at room temperature for 1 h in the dark, and stained with DAPI solution for 10 min. Additionally, the primary antibody against MMP-9 was incubated with the slides at 4 °C overnight to assess the metastasis of melanoma, which were washed with PBS twice and treated with the secondary antibody at room temperature for 1 h. Finally, the slides were incubated with diaminobenzidine chromogen (DAB) for 5 min, counterstained with haematoxylin, dehydrated and mounted. For the in situ apoptosis of metastatic melanoma, the TUNEL staining was performed at 37 °C for 1 h according to the manufacturer's protocol. The slides were stained with DAPI solution at room temperature for 10 min, and the stained sections were observed by IX73P1F fluorescence microscopy (Olympus, Tokyo, Japan).

2.13. Biocompatibility analysis of porous PLGA microspheres

At the end of the therapeutic treatment, the main organs (heart, liver, spleen and kidney) and the blood of mice were harvested. The organs were fixed with 4% (v/v) PFA and applied for histopathological analysis by H&E staining. The blood samples of mice were placed for 1 h and centrifugated at 3,000 r/min for 15 min to collect the serum. The content of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN) and creatinine (CRE) in serum was measured by automatic biochemical analyzer (Cheray 800, Shenzhen, China).

2.14. Statistical analysis

Data were presented as mean value ± standard deviation (SD) of triplicate experiments. The comparison of two groups was statistically analyzed using student's t-test in SPSS 24.0 (IBM, Armonk, NY), and data were considered as statistical significance (*P < 0.05, **P < 0.01).

3. Results and discussion

3.1. Preparation and characterization of porous PLGA microspheres

Double emulsification/solvent evaporation method was employed to prepare porous PLGA microspheres including blank microspheres (blank), ICA-loaded microspheres (PPM-1), miR-23b-loaded microspheres (PPM-2), and microspheres harboring ICA and miR-23b (PPM-3), as shown in Fig. 1A. The morphology of porous PLGA microspheres was detected by SEM (Fig. 1B). Smooth and spherical structure with uniform pores could be clearly observed for all these microspheres. The porosity of microspheres was caused by the decomposition of porogen ammonia bicarbonate into ammonia and carbon dioxide during homogenization. The particle size, zeta potential, drug loading and encapsulation efficiency of porous PLGA microspheres were measured (Table 1). Compared to blank microspheres, the zeta potential of PPM-1 increased attributing to the successful loading of ICA in microspheres. Similar particle size has been found for both blank and PPM-1 microspheres, suggesting that the encapsulation of ICA did not change the geometric structure of microspheres. To realize the loading and delivery of miR-23b, cationic carrier PEI was used to condense miR-23b plasmid to prepare nanoparticles, with particle size and zeta potential of 259.5 ± 45.2 nm and 11.3 ± 0.1 mV, respectively. Meanwhile, the addition of PEI was favorable for maintaining the integrity and biological activity of plasmids during freezing or spraying. After the encapsulation of PEI/miR-23b nanoparticles into porous PLGA microspheres, zeta potential of PPM-2 significantly decreased (−1.5 ± 0.2 mV) owing to the shielding of positively charged PEI by PLGA, which was beneficial to minimize the risk of inflammation response in the lung tissue since the positively charged particles induced the production of inflammatory cytokines after pulmonary delivery [32]. Slightly larger particle size could be detected in PPM-2 and PPM-3 microspheres than blank microspheres (22.3 ± 5.8 and 20.8 ± 4.7 µm). The aerodynamic diameter was a key parameter of microspheres administered in an inhalation route. All these PLGA microspheres showed aerodynamic diameter values of 4–5 µm, which was essential to realize the efficient deposition of particles in the lung alveolar [33]. Hence, these results indicated that when PLGA microspheres were well aerosolized and properly inhaled, they could be readily accumulated in the deep region of lung tissue. Also, the highly porous structure endowed the microspheres with more hydrophobic space for the encapsulation of ICA, with drug loading and encapsulation efficiency of 1.98% ± 0.04% and 81.36% ± 1.54% for PPM-1, respectively. In PPM-2, the miR-23b loading and encapsulation efficiency were measured to be 0.13‰ ± 0.01‰ and 80.67% ± 2.64%. Compared with PPM-1 and PPM-2, PPM-3 exhibited the slight decrease of drug loading and encapsulation efficiency (ICA, drug loading and encapsulation efficiency of 1.81% ± 0.08% and 78.11% ± 3.51%; miR-23b, drug loading and encapsulation efficiency of 0.12‰ ± 0.01‰ and 73.79% ± 3.32%), suggesting that the co-delivery of ICA and miR-23b might affect the drug loading process.

Fig 1 — Preparation and characterization of porous PLGA microspheres. (A) Schematic diagram for the preparation of porous PLGA microspheres harboring ICA and miR-23b by double emulsification/solvent evaporation method. (B) SEM images of different porous PLGA microspheres. Scale bar: 100 µm. (C) Cumulative release of ICA and miR-23b from PPM-1, PPM-2 and PPM-3 in Gamble solution. Data were presented as mean value ± SD of triplicate experiments.

Table 1.

Particle size, zeta potential, aerodynamic diameter, drug loading and encapsulation efficiency of different porous PLGA microspheres.

Sample	Zeta potential (mV)	Particle size (µm)	Aerodynamic diameter (µm)	DL for icariin (%, w/w)	DL for miR-23b (‰, w/w)	EE for icariin (%)	EE for miR-23b (%)
PEI/miR-23b	11.3 ± 0.1	259.5 ± 45.2 nm	–	–	–	–	–
Blank	−9.0 ± 0.4	18.8 ± 3.9	4.3 ± 0.8	–	–	–	–
PPM-1	−4.7 ± 0.1	18.4 ± 4.7	3.8 ± 1.1	1.98 ± 0.04	–	81.36 ± 1.54	–
PPM-2	−1.5 ± 0.2	22.3 ± 5.8	4.8 ± 0.2	–	0.13 ± 0.01	–	80.67 ± 2.64
PPM-3	−1.8 ± 0.1	20.8 ± 4.7	4.1 ± 0.1	1.81 ± 0.08	0.12 ± 0.01	78.11 ± 3.51	73.79 ± 3.32

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Data were presented as mean value ± SD, n = 3.

Further, the in vitro drug release profiles of PPM-1, PPM-2 and PPM-3 were monitored in Gamble solution at pH 7.4 (a simulation of lung fluid). As shown in Fig. 1C, ICA and miR-23b were gradually released from porous PLGA microspheres, and the release was almost complete until 8 d, indicating that the microspheres did not lead to a burst release. The sustained release manner was mainly attributed to the suitable porosity of microspheres and the gradual degradation of PLGA. PPM-3 showed similar release of ICA to PPM-1, but slightly lower release of miR-23b was observed in PPM-3 than PPM-2. The phenomenon was probably caused by that the ICA loading partially obstructed the pores of microspheres, thereby hindering the diffusion of solution. Particularly, the cumulative release amount of ICA and miR-23b reached 87.06% and 59.28% in PPM-3 over 8-d period. Meanwhile, miR-23b was released from porous PLGA microspheres in the form of PEI/miR-23b nanoparticles (Fig. S1).

3.2. Cellular uptake of released components from porous PLGA microspheres

To assess the cellular uptake of components released from porous PLGA microspheres, PEI was labeled by RBITC to form the fluorescent PEI/miR-23b nanoparticles and the fluorescent dye coumarin-6 was used as a hydrophobic drug model to replace ICA. As shown in Fig. S2, both green and red fluorescence could be observed in PPM-3, demonstrating that coumarin-6 and RBITC-labeled PEI/miR-23b nanoparticles were successfully encapsulated and uniformly distributed in porous PLGA microspheres. The release solutions were then collected from porous PLGA microspheres in Gamble solution at pH 7.4, and used to incubate with A549 cells for 48 h. As shown in Fig. 2A, compared with the untreated cells, green and red fluorescence could be clearly observed in the cells after the treatment with the release solutions from PPM-1 and PPM-2, respectively. Dual fluorescence could be simultaneously detected for the cells treated with the release solution from PPM-3. These results suggested that coumarin-6 and RBITC-labeled PEI/miR-23b nanoparticles were effectively released from PLGA microspheres and internalized by the cells. Further, the fluorescent intensity was analyzed by flow cytometry (Fig. 2B), which also elucidated the successful uptake of coumarin-6 and RBITC-labeled PEI/miR-23b nanoparticles in A549 cells. Relative miR-23b level analysis showed that in comparison to blank and PPM-1 groups, higher miR-23b expression could be observed in the cells treated with the release solution from PPM-2 (Fig. S3). Notably, the miR-23b content in PPM-3 group was higher than that in PPM-2 group, suggesting that the combination of ICA and PEI/miR-23b was beneficial for up-regulating the miR-23b level and obtaining better anti-tumor efficacy.

Fig 2 — Cellular uptake of coumarin-6 and RBITC-labeled PEI/miR-23b nanoparticles in the release solutions from porous PLGA microspheres, in which the incubation with A549 cells were conducted at 37 °C for 48 h. (A) CLSM images of coumarin-6 (green) and RBITC-labeled PEI/miR-23b (red) in A549 cells. Nucleus (blue) was stained by Hoechst 33342. Scale bar: 20 µm. (B) Flow cytometric analysis of coumarin-6 (a) and RBITC (b) signals of A549 cells after the treatment with release solutions from different PLGA microspheres. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.3. In vitro anti-proliferative effect

The in vitro anti-proliferative effect of release solutions from porous PLGA microspheres was detected in A549 cells by MTT assay. As shown in Fig. 3A, compared with blank group, the release solutions from other microspheres could inhibit the cell proliferation, e.g., cell viability of 43.34% and 76.20% after the treatment with release solutions from PPM-1 and PPM-2 on Day 8, respectively. These results demonstrated that both ICA and PEI/miR-23b nanoparticles could execute the anti-tumor function, and the release solutions containing ICA possessed stronger anti-proliferative effect than those from PPM-2. The most significant proliferative inhibition was achieved in the cells treated with the release solution from PPM-3 on Day 8 (cell viability of 33.54%), owing to the collective tumor suppression of ICA and PEI/miR-23b nanoparticles. The release solutions on Day 8 were applied in live/dead staining assay to further evaluate the anti-proliferative effect. As illustrated in Fig. S4, compared with the control and blank groups, clear red fluorescence could be observed after the treatment with the release solutions from PPM-1, PPM-2 and PPM-3, indicating the successful induction of anti-proliferative effect. Particularly, the co-delivery of ICA and PEI/miR-23b nanoparticles triggered the strongest anti-proliferative efficiency. Similarly, the release solution from PPM-3 microspheres at Day 8 displayed the highest capability to inhibit the formation of cell colonies (Fig. S5).

Fig 3 — *In vitro* anti-proliferative effect of release solutions from porous PLGA microspheres. (A) Cell viability of A549 cells treated by release solutions from porous PLGA microspheres at different days. (B) The *in situ* cell apoptosis analysis of A549 cells by TUNEL staining. Scale bar: 100 µm. (C) Cell apoptosis analysis of A549 cells treated by release solutions from porous PLGA microspheres. (D) Cell cycle arrest analysis of A549 cells treated by release solutions from porous PLGA microspheres and (E) the relative distribution of cell population in different cell cycle phases. (a) Control, (b) blank, (c) PPM-1, (d) PPM-2 and (e) PPM-3.

3.4. In vitro apoptosis and cell cycle arrest analysis

To reveal the mechanism underlying anti-proliferative action of release solutions from PLGA microspheres, cell apoptosis and cell cycle arrest were analyzed in A549 cells after the treatment with different release solutions. First, TUNEL staining assay was conducted to detect the cell apoptosis (Fig. 3B). Compared to blank group, green fluorescence could be clearly observed in the groups of release solutions from PPM-1 and PPM-2, indicating that both ICA and PEI/miR-23b nanoparticles could trigger the cell apoptosis. Moreover, the number of TUNEL-positive cells increased when the cells were incubated with the release solution from PPM-3, meaning the stronger apoptotic effect. Flow cytometric analysis was then used to detect the cell apoptosis induced by the release solutions, based on Annexin V-FITC/PI staining (Fig. 3C). Compared with untreated and blank groups, significant apoptotic effect was triggered in the cells treated with the release solutions from PPM-1, PPM-2 and PPM-3, in which the highest proportion of apoptotic cells was achieved in PPM-3 group (early apoptotic ratio of 35.36%).

To investigate the mechanism of cell apoptosis induced by release solutions, the expression of apoptosis-related proteins was analyzed using Western blotting (Fig. 4 and S6). The expression of procaspase-3 was significantly reduced after the treatment with release solutions from porous PLGA microspheres, implying that both ICA and miR-23b could activate caspase-3 by the cleavage of procaspase-3. Meanwhile, the activity of caspase-3 significantly increased when the cells were exposed to release solutions (Fig. S7). Moreover, the cleavage band of poly(ADP-ribose) polymerase (PARP) could be clearly observed after the treatment with release solutions from PPM-1 and PPM-3 owing to the role of ICA. Caspase-3 has been identified as the primary executor of apoptosis which leads to the cleavage of downstream proteins in apoptotic pathway including PARP [34]. These findings indicated that ICA and miR-23b could activate capase-3 and further produce cleaved PARP to execute the induction function of cell apoptosis. Besides, the expression of procaspase-8 and procaspase-9 was both downregulated in PPM-1, PPM-2 and PPM-3 groups, suggesting that both caspase-8 and caspase-9 were activated by the cleavage of their precursors. Consistently, the relative activities of caspase-8 and caspase-9 were elevated in PPM-1, PPM-2 and PPM-3 groups (Fig. S7), revealing that the delivery of ICA and miR-23b by porous PLGA microspheres could activate both death receptor-mediated pathway and mitochondrial-dependent pathway to induce cell apoptosis. The loss of mitochondrial transmembrane potential could also trigger the apoptosis; thus, the mitochondrial membrane potential of cancer cells was detected by JC-1 probe. As shown in Fig. S8, the release solutions containing ICA and miR-23b induced the depletion of mitochondrial membrane potential as evidenced by higher conversion of JC-1 aggregates to monomers, thereby eliciting the mitochondrial-dependent apoptosis pathway.

Fig 4 — Western blotting analysis for the expression level of key proteins in A549 cells after the treatment with release solutions from different porous PLGA microspheres. Lane 1: control, lane 2: blank, lane 3: PPM-1, lane 4: PPM-2 and lane 5: PPM-3.

Furthermore, the expression levels of Survivin and Bcl-2 significantly decreased in A549 cells after the treatment with release solutions from PPM-1, PPM-2 and PPM-3 (Fig. 4). Survivin has been considered as an inhibitor of caspase members, leading to the anti-apoptotic effect in cells, and Bcl-2 could block the cell apoptosis by the preservation of mitochondrial membrane integrity [35,36]. The elevated expression of Bcl-2 and Survivin has been accepted to be predictive factor in the chemoresistance of lung cancers [37]. Thus, the suppression of Bcl-2 and Survivin by release solutions containing ICA and miR-23b mitigated the chemoresistance of cancer cells, thereby strengthening the cell apoptotic response. Moreover, the PTEN expression in A549 cells was detected since PTEN is an essential tumor suppressor regulating the cell proliferation and survival [38]. As shown in Fig. 4, increased expression of PTEN could be clearly observed after the treatment with release solutions containing ICA and miR-23b, thereby inducing the cell apoptosis.

In addition to apoptosis, cell cycle arrest is considered as another pivotal factor in the anti-proliferative effect. As shown in Figs. 3D and 3E, compared with blank group, the release solution from PPM-1 arrested the cell cycle at G1 phase with G1 ratio of 77.26%. In contrast, the percentage of cells in the G2 phase significantly decreased while the cell distribution in S phase increased after the incubation with the release solution from PPM-2, suggesting that miR-23b could achieve the cell cycle arrest at S phase with ratio of 28.26%. Intriguingly, the co-delivery of ICA and PEI/miR-23b nanoparticles showed an obvious cell arrest at G1 phase (G1 phase of 74.65%), which was probably caused by more efficient internalization of ICA. Moreover, as shown in Fig. 4, the release solution from PPM-3 microspheres downregulated the expression of Cyclin D1, since its expression was associated with the G1 transition [39,40]. Taken together, the co-delivery of ICA and miR-23b by porous PLGA microspheres could be a promising strategy to induce the cell apoptosis and cell cycle arrest, leading to the desirable anti-proliferative effect in lung cancer treatment.

3.5. Inhibition of cell migration and invasion

The wound healing and Transwell migration assays were conducted to assess the inhibition of tumor metastasis by the released solutions. As shown in Fig. 5, the cells treated with the release solution from blank microspheres constantly migrated to the scratch area, while the release solutions from PPM-1 and PPM-2 inhibited the migration of cells to the scratch, with wound healing ratios of 81.07% and 75.21% after 72 h, respectively. Particularly, the co-delivery of ICA and PEI/miR-23b nanoparticles retarded the migratory cells to the scratched zones more efficiently than PPM-1 and PPM-2 groups. Further, the invasive ability of A549 cells was examined after the treatment with release solutions (Fig. S9). Similarly, compared with blank group, the release solutions from drug/gene-loaded PLGA microspheres could inhibit the infiltration of cells, in which the fewest cells migrating on the bottom layer of Transwell membrane were found in PPM-3 group. In the process of cell metastasis, MMP-9 modulates the metastatic cascade and drives the progression of cell migration and invasion, whereas Notch-1 promotes metastatic behavior of cancer cells by regulating the initiation of metastasis [41,42]. As shown in Fig. 4, the release solution from PPM-3 group inhibited the expression of MMP-9 and Notch-1, thus blocking the invasion and migration of cancer cells. Overall, the co-delivery of ICA and miR-23b by porous PLGA microspheres improved the capability to prevent the metastatic progression of lung cancer.

Fig 5 — Wound healing assay (A) and the quantitative wound size (B) of A549 cells after the treatment with release solutions from porous PLGA microspheres. Data were presented as mean value ± SD (n = 3, **P < 0.01). Scale bar: 200 µm.

3.6. In vivo biodistribution of porous PLGA microspheres via pulmonary delivery

Owing to the respiratory barriers, the mucociliary escalator's clearance and the capture of alveolar macrophages in the lung tissue, it is still a great challenge in pulmonary drug delivery. To improve the lung deposition of drugs, porous PLGA microspheres were lyophilized as an inhalable dry powder as this formulation exhibited practical advantages over the liquid aerosol in terms of improved stability, ease of operation, and convenience of storage and transportation. As shown in Fig. S10A, the aerosolization of porous PLGA microspheres was accomplished in 0.06 s after the actuation, indicating that the microspheres presented desirable mobility for aerosolization. Subsequently, the dry powder formulations of PPM-1, PPM-2 and PPM-3 microspheres were administered to healthy BALB/C mice via intratracheal route by a Penn-Century DP-4 dry powder insufflator (Fig. S10B and Supplementary video).

The in vivo biodistribution of porous PLGA microspheres were then evaluated at multiple time points. As visualized in Fig. 6A, obvious fluorescence of coumarin-6 and RBITC-labeled PEI/miR-23b nanoparticles could be immediately detected in lungs after the inhalation of PPM-1, PPM-2 and PPM-3 microspheres. These results indicated that porous PLGA microspheres realized a favorable deposition in lung lobes and benefited the drug accumulation in the local tissue, owing to their desirable aerodynamic diameter. The respiratory tract exerts the critical function to filter and separate particles from the air, in which large particles with an aerodynamic diameter greater than 6 µm were easily deposited in the upper airway while the inhaled particles smaller than 1 µm were difficult to reach the lung tissue due to Brownian motion [43]. The optimal aerodynamic diameter in the range of 1–5 µm was essential to achieve the lung deposition of particles, and thus porous PLGA microspheres in the present study could fulfill the requirement in the lung deposition after inhalation. Besides, the fluorescence of coumarin-6 and RBITC remained stable over 48 h and lasted for 10 d, showing that porous PLGA microspheres released the cargoes in a sustainable manner and prolonged the drug retention in lung tissues. Though ICA as a lipophilic drug may suffer from the mucociliary clearance in the airway, PLGA microspheres protected it from the rapid elimination in the airway by efficient lung deposition. More importantly, rational development of the nanoparticles-loaded microparticles was mutually beneficial since porous PLGA microspheres realized the deep deposition of nanoparticles in lungs and protected them from the macrophages’ capture, and subsequently the released nanoparticles could promote the miR-23b transfection in the tumor cells. For a further biodistribution analysis of porous PLGA microspheres, the lung and non-lung compartments were excised to quantify the fluorescence intensity (Fig. 6B and S11). The fluorescence intensities of coumarin-6 and RBITC in the non-lung organs were much lower than lungs, in which weak fluorescence of coumarin-6 and RBITC was detected in liver and kidney after 48 h post-administration. The phenomenon suggested that the components released from porous PLGA microspheres were leaked by the circulating blood system and uptake by the cells of reticuloendothelial system recruited by other organs.

3.7. Anti-tumor efficacy of porous PLGA microspheres by pulmonary delivery

The anti-tumor efficacy of PPM-1, PPM-2 and PPM-3 microspheres was evaluated in the mice model bearing B16F10 melanoma-metastatic lung cancer. As shown in Fig. 7A, the pulmonary administration of different microspheres was performed by intratracheal route on Day 7, 14 and 21 after the implantation of B16F10 cells. On Day 28, the lung tissues of tumor-bearing mice were excised to evaluate the therapeutic efficacy of different formulations. As shown in Fig. 7B, the lungs excised from healthy mice showed normal morphology, whereas the lungs of melanoma-bearing mice were occupied by the melanoma nodules. In contrast, the tumor growth was obviously inhibited in PPM-1, PPM-2 and PPM-3 groups, in terms of lower lung weight and less tumor nodes in the lung, suggesting that ICA or miR-23b could inhibit the growth and metastasis of tumors (Fig. 7C and 7D). Particularly, the best anti-cancer efficacy was realized in the co-delivery of ICA and miR-23b by PPM-3 microspheres, as evidenced by the smallest area of tumor nodules formed on the lung. Moreover, the average lung weight of mice treated with PPM-3 (0.30 g) was much lower than those in PPM-1 (0.52 g) and PPM-2 (0.74 g), confirming that the co-delivery of these two components exhibited superior ability to inhibit the tumor progression. Further, another batch of animal experiment was conducted to evaluate the anti-tumor efficacy of porous PLGA microspheres by pulmonary delivery, in which the inoculation number of B16F10 cells was 5.0 × 10⁵ for each mouse, and the intervention time was extended to five weeks (Fig. S12). Similarly, lung image observation, lung weight and nodule area revealed that PPM-3 microspheres showed a pronounced inhibitory effect on the tumor proliferation and metastasis (Fig. S13). These results demonstrated that porous PLGA microspheres harboring ICA or miR-23b could be accumulated in the lung tissues through pulmonary delivery and further released the cargoes to elicit the effective inhibition of tumor growth and metastasis.

Fig 7 — *In vivo* anti-tumor efficacy of different microspheres via inhalation administration. (A) The therapeutic scheme of porous PLGA microspheres using BALB/c mice with melanoma-metastatic lung cancer. (B) Photographs of lungs obtained from the mice after the treatment with different PLGA microspheres. (C) Average lung weight obtained from the lungs with metastatic tumors. (D) Relative area of melanoma nodules. Data were presented as mean value ± SD (n = 4, *n.d.* not determined, *P < 0.05, **P < 0.01).

To assess the therapeutic efficacy of different microspheres after inhalation, the histology of lungs from tumor-bearing mice was examined by H&E staining. As demonstrated in Fig. 8, compared with the healthy mice, the absence of alveolar structure and many visible pigmentations and melanomas could be observed in the lung tissues of mice treated with blank microspheres, indicating that the metastatic tumors completely occupied the lung lobe. In contrast, the number of tumor pigmentations in the lung tissues decreased when the mice were administered via the inhalation of PPM-1, PPM-2 and PPM-3 microspheres, in which more typical structure of alveolar lumen could be observed in PPM-3 group than PPM-1 and PPM-2. Further, the proliferation of tumor cells was detected by immunofluorescence analysis of Ki-67 and TUNEL (Fig. 8). Ki-67 is one of key biomarkers of various malignant tumors as it is expressed during all stages of cell cycle [44]. Compared with the healthy mice, increased Ki-67 expression could be observed in the untreated tumor-bearing mice. The number of Ki-67-positive cells decreased in the mice after the treatment with PPM-1, PPM-2 and PPM-3, demonstrating that the inhalation of drug/gene-loaded porous PLGA microspheres could suppress the proliferation of tumor cells. Notably, PPM-3 group exhibited the smallest proportion of Ki-67-positive cells among these formulations. In addition, compared to blank microspheres, PPM-1, PPM-2 and PPM-3 induced obvious apoptosis in the tumor tissue, as evidenced by the strong green fluorescence of TUNEL signal. Besides the anti-proliferative effect, we also detected whether the anti-metastatic response was associated with the decreased MMP-9 expression in tumor tissues. Clearly, compared with the healthy tissue, the MMP-9 expression was improved in the tumor-bearing mice while the inhalation of porous PLGA microspheres attenuated its expression, leading to the anti-metastasis of tumor.

Fig 8 — H&E staining, immunofluorescence analysis of Ki-67 and TUNEL, and IHC analysis of MMP-9 expression in the melanoma-metastatic lung tissues after the treatment with different porous PLGA microspheres. Black scale bar: 100 µm; white scale bar: 20 µm.

3.8. Biocompatibility of porous PLGA microspheres

To evaluate the biocompatibility of porous PLGA microspheres, body weight, serum biochemical analysis and histological assessment were carried out at the end of treatment. The changes of body weight were monitored every 4 d during the treatment to access the systemic safety of microspheres. The body weight of tumor-implanted mice gradually decreased compared with the healthy mice (Fig. S14). In contrast, the body weight did not change in the tumor-bearing mice treated with drug/gene-loaded microspheres, in which the mice in PPM-3 group showed comparable body weight to the healthy mice. Moreover, since ICA and miR-23b released from porous PLGA microspheres were partially distributed in the liver and kidney tissues after inhalation, serum biochemical analysis was conducted to evaluate the liver and kidney function by measuring the content of ALT, AST, ALP, CRE and BUN. As shown in Fig. S15, there were no significant differences in serum biochemical parameters between healthy mice and the groups treated with porous PLGA microspheres, indicating that all porous PLGA microspheres had negligible influences on the renal and hepatic function. Finally, the main organs including heart, liver, spleen and kidney were collected and sectioned to examine the organic lesion induced by porous PLGA microspheres (Fig. S16). Compared with the healthy group, the main organs from the mice treated with different microspheres exhibited no significant morphological changes in terms of tissue degeneration, inflammation or necrosis. All these results demonstrated that the inhalation of porous PLGA microspheres harboring ICA and/or miR-23b possessed desirable compatibility.

4. Conclusion

In the present study, porous PLGA microspheres harboring ICA and PEI/miR-23b nanoparticles were successfully prepared for the treatment of lung cancer in an inhalation route. The microspheres possessed uniform and porous morphology, and exhibited unique characteristics including desirable aerodynamic diameter, high drug encapsulation and sustained drug release profile. The released ICA and PEI/miR-23b nanoparticles from microspheres could be easily uptake by lung cancer cells, contributing to the anti-proliferative and anti-metastatic effect. After the proper aerolization, porous PLGA microspheres could be well inhaled and deposited in the lungs, thereby obtaining favorable therapeutic efficacy in the mice bearing melanoma-metastatic lung cancer. Finally, the microspheres were demonstrated to possess good biocompatibility after the inhalation. Overall, porous PLGA microspheres could be served as a promising carrier for the inhalable delivery of drugs and genes in the treatment of lung cancers and other pulmonary diseases.

Conflict of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Acknowledgments

The authors gratefully acknowledge the supports from the National Natural Science Foundation of China (32271319 and 32071267), the Science and Technology Department of Jilin Province (YDZJ202301ZYTS537 and 20240402035GH), the Development and Reform Commission of Jilin Province (2023C015), and the “Medicine + X” cross-innovation team of Bethune Medical Department of Jilin University “Leading the Charge with Open Competition” construction project (2022JBGS04).

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.ajps.2024.101008. The figures and tables with “S“ before the serial number are included in the Supplementary material.

Contributor Information

Haobo Han, Email: hanhaobo@jlu.edu.cn.

Quanshun Li, Email: quanshun@jlu.edu.cn.

Appendix. Supplementary materials

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Download video file^{(2MB, mp4)}

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PERMALINK

Porous PLGA microspheres for the inhalation delivery of icariin and miR-23b in the treatment of metastatic lung cancer

Boyu Xiong

Xinxin Shao

Guangxu Fang

Mengmeng Dong

Haobo Han

Quanshun Li

Abstract

Graphical abstract

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Preparation of porous PLGA microspheres

2.3. Characterization of porous PLGA microspheres

2.4. Drug loading and encapsulation efficiency measurement

2.5. Cellular uptake of the components released from porous PLGA microspheres

2.6. Anti-proliferative analysis

2.7. Cell apoptosis and cell cycle arrest analysis

2.8. Western blotting analysis

2.9. Wound healing assay

2.10. In vivo biodistribution of porous PLGA microspheres

2.11. In vivo anti-tumor efficacy of porous PLGA microspheres

2.12. Immunohistochemical (IHC) analysis of metastatic melanoma

2.13. Biocompatibility analysis of porous PLGA microspheres

2.14. Statistical analysis

3. Results and discussion

3.1. Preparation and characterization of porous PLGA microspheres

Fig. 1.

Table 1.

3.2. Cellular uptake of released components from porous PLGA microspheres

Fig. 2.

3.3. In vitro anti-proliferative effect

Fig. 3.

3.4. In vitro apoptosis and cell cycle arrest analysis

Fig. 4.

3.5. Inhibition of cell migration and invasion

Fig. 5.

3.6. In vivo biodistribution of porous PLGA microspheres via pulmonary delivery

Fig. 6.

3.7. Anti-tumor efficacy of porous PLGA microspheres by pulmonary delivery

Fig. 7.

Fig. 8.

3.8. Biocompatibility of porous PLGA microspheres

4. Conclusion

Conflict of interest

Acknowledgments

Footnotes

Contributor Information

Appendix. Supplementary materials

References

Associated Data

Supplementary Materials

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