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Targeted Oral Delivery of Paclitaxel Using Colostrum-Derived Exosomes

Kandimalla, Raghuram, Aqil, Farrukh, Alhakeem, Sara, Jeyabalan, Jeyaprakash, Tyagi, Neha, Agrawal, Ashish, Yan, Jun, Spencer, Wendy, Bondada, Subbarao, Gupta, Ramesh·2021·Cancers
DOI 10.3390/cancers13153700PMC PMC8345039
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Abstract

Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small-cell lung cancer (NSCLC) is the most common type accounting for 84% of all lung cancers. Paclitaxel (PAC) is a widely used drug in the treatment of a broad spectrum of human cancers, including lung. While efficacious, PAC generally is not well tolerated and its limitations include low aqueous solubility, and significant toxicity. To overcome the dose-related toxicity of solvent-based PAC, we utilized bovine colostrum-derived exosomes as a delivery vehicle for PAC for the treatment of lung cancer. Colostrum provided higher yield of exosomes and could be loaded with higher amount of PAC compared to mature milk. Exosomal formulation of PAC (ExoPAC) showed higher antiproliferative activity and inhibition of colony formation against A549 cells compared with PAC alone, and also showed antiproliferative activity against a drug-resistant variant of A549. To further enhance its efficacy, exosomes were attached with a tumor-targeting ligand, folic acid (FA). FA-ExoPAC given orally showed significant inhibition (>50%) of subcutaneous tumor xenograft while similar doses of PAC showed insignificant inhibition. In the orthotopic lung cancer model, oral dosing of FA-ExoPAC achieved greater efficacy (55% growth inhibition) than traditional i.v. PAC (24–32% growth inhibition) and similar efficacy as i.v. Abraxane (59% growth inhibition). The FA-ExoPAC given i.v. exceeded the therapeutic efficacy of Abraxane (76% growth inhibition). Finally, wild-type animals treated with p.o. ExoPAC did not show gross, systemic or immunotoxicity. Solvent-based PAC caused immunotoxicity which was either reduced or completely mitigated by its exosomal formulations. These studies show that a tumor-targeted oral formulation of PAC (FA-ExoPAC) significantly improved the overall efficacy and safety profile while providing a user-friendly, cost-effective alternative to bolus i.v. PAC and i.v. Abraxane.

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Figure 1 Characterization and drug loading of colostrum-derived exosomes. Size, polydispersity index (PDI), and zeta potential (ZP) of exosomes, FA-Exo, ExoPAC and FA-ExoPAC, analyzed by Zetasizer. Data represent mean ± SD from three preparations (A). Analysis of exosomes and ExoPAC by atomic force microscopy (AFM) after diluting with deionized water up to 10 μg/mL. For measurement, samples were placed on a silica wafer and air-dried for 30 min. AFM in tapping mode and aluminum-coated silicon probes were used for imaging (B). The bar diagram shows the quenching of autofluorescence from the exosomes following PAC loading (C). Higher quenching of fluorescence in the presence of higher drug load suggests a hydrophobic interaction of drug with exosomal proteins.
Figure 1 Characterization and drug loading of colostrum-derived exosomes. Size, polydispersity index (PDI), and zeta potential (ZP) of exosomes, FA-Exo, ExoPAC and FA-ExoPAC, analyzed by Zetasizer. Data represent mean ± SD from three preparations (A). Analysis of exosomes and ExoPAC by atomic force microscopy (AFM) after diluting with deionized water up to 10 μg/mL. For measurement, samples were placed on a silica wafer and air-dried for 30 min. AFM in tapping mode and aluminum-coated silicon probes were used for imaging (B). The bar diagram shows the quenching of autofluorescence from the exosomes following PAC loading (C). Higher quenching of fluorescence in the presence of higher drug load suggests a hydrophobic interaction of drug with exosomal proteins.CC BY · PubMed Central
Figure 2 ExoPAC inhibits proliferation of drug-sensitive and drug-resistant cells. (A) Drug-sensitive (A549) and drug-resistant (A549TR) cells were treated with Exo, ExoPAC and FA-ExoPAC and compared with Abraxane. Antiproliferative activity was determined by MTT assay after 72 h. Exosomal PAC dose-dependently inhibited the proliferation of drug-sensitive A549 (A) and drug-resistant A549TR cells (B).
Figure 2 ExoPAC inhibits proliferation of drug-sensitive and drug-resistant cells. (A) Drug-sensitive (A549) and drug-resistant (A549TR) cells were treated with Exo, ExoPAC and FA-ExoPAC and compared with Abraxane. Antiproliferative activity was determined by MTT assay after 72 h. Exosomal PAC dose-dependently inhibited the proliferation of drug-sensitive A549 (A) and drug-resistant A549TR cells (B).CC BY · PubMed Central
Figure 3 Exosomal PAC inhibited colony formation in NSCLC cells. Representative images showing the colony formation assay in drug-sensitive A549 (A) and drug-resistant A549TR (B) cells. Lung cancer cells were seeded (500 cells/well) in a six-well plate and incubated with different concentrations of PAC and ExoPAC. After 10 days, developed colonies were fixed, stained and counted manually. While PAC was effective, only against drug-sensitive cells (A1,B1), ExoPAC shows dose-dependent inhibition of colony formation of both sensitive and resistant cells (A2,B2). Statistical analysis was performed using the Student’s t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 3 Exosomal PAC inhibited colony formation in NSCLC cells. Representative images showing the colony formation assay in drug-sensitive A549 (A) and drug-resistant A549TR (B) cells. Lung cancer cells were seeded (500 cells/well) in a six-well plate and incubated with different concentrations of PAC and ExoPAC. After 10 days, developed colonies were fixed, stained and counted manually. While PAC was effective, only against drug-sensitive cells (A1,B1), ExoPAC shows dose-dependent inhibition of colony formation of both sensitive and resistant cells (A2,B2). Statistical analysis was performed using the Student’s t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.CC BY · PubMed Central
Figure 4 Antitumor activity against subcutaneous xenografts. Following inoculation with A549 cells, nude mice were treated with oral gavage three times a week with PAC (6 mg/kg bw), ExoPAC and FA-ExoPAC (6 mg PAC and 50 mg Exo protein/kg bw). Data represent average ± SD of means (n = 8). Statistical analysis was performed using the Student’s t-test. * p < 0.05; ** p < 0.01.
Figure 4 Antitumor activity against subcutaneous xenografts. Following inoculation with A549 cells, nude mice were treated with oral gavage three times a week with PAC (6 mg/kg bw), ExoPAC and FA-ExoPAC (6 mg PAC and 50 mg Exo protein/kg bw). Data represent average ± SD of means (n = 8). Statistical analysis was performed using the Student’s t-test. * p < 0.05; ** p < 0.01.CC BY · PubMed Central
Figure 5 Antitumor activity against orthotopic xenografts. Detection of orthotopic lung cancer using bioluminescent A549-Red-luc cells. (A) A1: image of live animals after 27 d of inoculation with 2 × 106 cells (3 mice/group). A2: Mean of bioluminescence signals. (B) Inhibition of A549 orthotopic lung tumors in NOD Scid female mice (n = 10) by i.v. paclitaxel (PAC), i.v. Abraxane, and p.o. ExoPAC and p.o. FA-ExoPAC (three doses weekly), all given at 6 mg/kg. FA-Exo was used as control for FA-ExoPAC. (C) Inhibition of A549 orthotopic lung tumors in NOD Scid female mice (n = 10) by i.v. PAC, Abraxane, FA-ExoPAC (once weekly) and p.o. ExoPAC and FA-ExoPAC (three doses weekly), all given at 4 mg/kg until three wks, then switched to 8 mg/kg, as indicated by an arrow in C2. Representative images of animals at different time points in the indicated treatment groups (C1) and time-dependent tumor inhibition (C2). Statistical analysis was performed using the Student’s t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 5 Antitumor activity against orthotopic xenografts. Detection of orthotopic lung cancer using bioluminescent A549-Red-luc cells. (A) A1: image of live animals after 27 d of inoculation with 2 × 106 cells (3 mice/group). A2: Mean of bioluminescence signals. (B) Inhibition of A549 orthotopic lung tumors in NOD Scid female mice (n = 10) by i.v. paclitaxel (PAC), i.v. Abraxane, and p.o. ExoPAC and p.o. FA-ExoPAC (three doses weekly), all given at 6 mg/kg. FA-Exo was used as control for FA-ExoPAC. (C) Inhibition of A549 orthotopic lung tumors in NOD Scid female mice (n = 10) by i.v. PAC, Abraxane, FA-ExoPAC (once weekly) and p.o. ExoPAC and FA-ExoPAC (three doses weekly), all given at 4 mg/kg until three wks, then switched to 8 mg/kg, as indicated by an arrow in C2. Representative images of animals at different time points in the indicated treatment groups (C1) and time-dependent tumor inhibition (C2). Statistical analysis was performed using the Student’s t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.CC BY · PubMed Central
Figure 6 Potential immunotoxicity of PAC and FA-ExoPAC. Female C57BL/6 mice were treated with Exo, PAC and FA-ExoPAC for four weeks. At euthanasia, spleen and bone marrow cells were collected. Live splenic (A) and bone marrow (B) cell counts were performed by trypan blue exclusion.
Figure 6 Potential immunotoxicity of PAC and FA-ExoPAC. Female C57BL/6 mice were treated with Exo, PAC and FA-ExoPAC for four weeks. At euthanasia, spleen and bone marrow cells were collected. Live splenic (A) and bone marrow (B) cell counts were performed by trypan blue exclusion.CC BY · PubMed Central
Figure 7 Potential immunotoxicity of PAC and FA-ExoPAC. Female C57BL/6 mice were treated with Exo, PAC and FA-ExoPAC for four weeks. At euthanasia, spleen and bone marrow cells were collected. Splenic cells were stained with multiple fluorochrome-conjugated antibodies. Samples were run using LSRII cytometer and data was analyzed by FlowJo software. Effect of treatment was analyzed on B cells, total T-cells, T helper cells, and cytotoxic T-cells (A), macrophages, neutrophils and dendritic cells (B), B cell (C), %LSK cells (D) and T-cell proliferation (E). All statistical analysis was performed by two-way ANOVA and compared with untreated control. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 7 Potential immunotoxicity of PAC and FA-ExoPAC. Female C57BL/6 mice were treated with Exo, PAC and FA-ExoPAC for four weeks. At euthanasia, spleen and bone marrow cells were collected. Splenic cells were stained with multiple fluorochrome-conjugated antibodies. Samples were run using LSRII cytometer and data was analyzed by FlowJo software. Effect of treatment was analyzed on B cells, total T-cells, T helper cells, and cytotoxic T-cells (A), macrophages, neutrophils and dendritic cells (B), B cell (C), %LSK cells (D) and T-cell proliferation (E). All statistical analysis was performed by two-way ANOVA and compared with untreated control. *, p < 0.05; **, p < 0.01; ***, p < 0.001.CC BY · PubMed Central
Figure 8 Potential immunotoxicity of PAC and FA-ExoPAC. Female C57BL/6 mice were treated with Exo, PAC and FA-ExoPAC for four weeks. At euthanasia, spleen and bone marrow cells were collected. Splenic cells were treated with the different stimulant for 24 h. Sups were collected and MesoScale analysis (V-PLEX of 6 cytokines) was performed. LPS was used for B-cell response and αCD3+αCD28 for T-cell response. All statistical analysis was performed by two-way ANOVA and compared with untreated control. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 8 Potential immunotoxicity of PAC and FA-ExoPAC. Female C57BL/6 mice were treated with Exo, PAC and FA-ExoPAC for four weeks. At euthanasia, spleen and bone marrow cells were collected. Splenic cells were treated with the different stimulant for 24 h. Sups were collected and MesoScale analysis (V-PLEX of 6 cytokines) was performed. LPS was used for B-cell response and αCD3+αCD28 for T-cell response. All statistical analysis was performed by two-way ANOVA and compared with untreated control. *, p < 0.05; **, p < 0.01; ***, p < 0.001.CC BY · PubMed Central

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Metadata

Authors
Kandimalla, R., Aqil, F., Alhakeem, S., Jeyabalan, J., Tyagi, N., Agrawal, A., Yan, J., Spencer, W., Bondada, S., & Gupta, R.
Venue
Cancers
Publisher
MDPI AG
Published
2021-07-23
Volume
13
Issue
15
Pages
3700
ISSN
2072-6694
DOI
10.3390/cancers13153700
PMC
PMC8345039
Cited by
99
References
3
Language
en
Update notices
correction (2021-07-23)
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
National Institutes of Health (R44-CA-221487)

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