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Active cargo loading into extracellular vesicles: Highlights the heterogeneous encapsulation behaviour

Chen, Chaoxiang, Sun, Mengdi, Wang, Jialin, Su, Liyun, Lin, Junjie, Yan, Xiaomei·2021·Journal of Extracellular Vesicles
DOI 10.1002/jev2.12163PMC PMC8558234
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Abstract

Extracellular vesicles (EVs) have demonstrated unique advantages in serving as nanocarriers for drug delivery, yet the cargo encapsulation efficiency is far from expectation, especially for hydrophilic chemotherapeutic drugs. Besides, the intrinsic heterogeneity of EVs renders it difficult to evaluate drug encapsulation behaviour. Inspired by the active drug loading strategy of liposomal nanomedicines, here we report the development of a method, named “Sonication and Extrusion‐assisted Active Loading” (SEAL), for effective and stable drug encapsulation of EVs. Using doxorubicin‐loaded milk‐derived EVs (Dox‐mEVs) as the model system, sonication was applied to temporarily permeabilize the membrane, facilitating the influx of ammonium sulfate solution into the lumen to establish the transmembrane ion gradient essential for active loading. Along with extrusion to downsize large mEVs, homogenize particle size and reshape the nonspherical or multilamellar vesicles, SEAL showed around 10‐fold enhancement of drug encapsulation efficiency compared with passive loading. Single‐particle analysis by nano‐flow cytometry was further employed to reveal the heterogeneous encapsulation behaviour of Dox‐mEVs which would otherwise be overlooked by bulk‐based approaches. Correlation analysis between doxorubicin auto‐fluorescence and the fluorescence of a lipophilic dye DiD suggested that only the lipid‐enclosed particles were actively loadable. Meanwhile, immunofluorescence analysis revealed that more than 85% of the casein positive particles was doxorubicin free. These findings further inspired the development of the lipid‐probe‐ and immuno‐mediated magnetic isolation techniques to selectively remove the contaminants of non‐lipid enclosed particles and casein assemblies, respectively. Finally, the intracellular assessments confirmed the superior performance of SEAL‐prepared mEV formulations, and demonstrated the impact of encapsulation heterogeneity on therapeutic outcome. The as‐developed cargo‐loading approach and nano‐flow cytometry‐based characterization method will provide an instructive insight in the development of EV‐based delivery systems.

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FIGURE 1 Preparation and analysis of Dox‐mEVs. (a) Schematic illustration representing the principle of SEAL. (b) Fluorescence analysis of Dox‐mEVs prepared by passive incubation and SEAL. (c) Fluorescence intensity of Dox‐mEVs at different ammonium sulfate concentration and sonication period (n = 3, mean ± SD). (d) Fluorescence intensity and particle size of Dox‐mEVs at various extrusion cycles (n = 3, mean ± SD). (e) Encapsulation efficiency and loading concentration of Dox‐mEVs at various drug input (n = 3, mean ± SD). (f) The encapsulation efficiency and particle size of Dox‐mEVs by various loading methods. p‐Values were calculated by t‐test. *p < 0.05, ****p < 0.0001 (n = 3, mean ± SD). (g) Drug release profile of free doxorubicin, as well as Dox‐mEVs prepared by SEAL and passive incubation, respectively (n = 3, mean ± SD). (h) TEM images of the unloaded mEVs (i) and SEAL‐prepared Dox‐mEVs (iii, scale bar = 100 nm). (ii) and (iv) depict a single mEV selected from the same image of (i) and (iii), respectively
FIGURE 1 Preparation and analysis of Dox‐mEVs. (a) Schematic illustration representing the principle of SEAL. (b) Fluorescence analysis of Dox‐mEVs prepared by passive incubation and SEAL. (c) Fluorescence intensity of Dox‐mEVs at different ammonium sulfate concentration and sonication period (n = 3, mean ± SD). (d) Fluorescence intensity and particle size of Dox‐mEVs at various extrusion cycles (n = 3, mean ± SD). (e) Encapsulation efficiency and loading concentration of Dox‐mEVs at various drug input (n = 3, mean ± SD). (f) The encapsulation efficiency and particle size of Dox‐mEVs by various loading methods. p‐Values were calculated by t‐test. *p < 0.05, ****p < 0.0001 (n = 3, mean ± SD). (g) Drug release profile of free doxorubicin, as well as Dox‐mEVs prepared by SEAL and passive incubation, respectively (n = 3, mean ± SD). (h) TEM images of the unloaded mEVs (i) and SEAL‐prepared Dox‐mEVs (iii, scale bar = 100 nm). (ii) and (iv) depict a single mEV selected from the same image of (i) and (iii), respectivelyCC BY-NC · PubMed Central
FIGURE 2 Single particle analysis of the drug loading capacity of SEAL‐prepared Dox‐mEVs by nFCM. (a) Representative SS and FL‐Dox burst traces of the Dox‐mEVs prepared by passive incubation (i, ii) and SEAL (iii, iv). (b) Bivariate dot plots of SS verse FL‐Dox of SEAL‐prepared Dox‐mEVs. (c) SS (i) and FL‐Dox (ii) intensity distribution histograms of the two subpopulations in SEAL‐prepared Dox‐mEVs. (d) Particle concentration and the active loading fraction of Dox‐mEVs prepared and purified by various methods. Note. The numbers on the above of each column indicated the active loading fractions. (e) The median SS (i) and FL‐Dox (ii) intensity of Dox‐mEVs prepared and purified by various methods
FIGURE 2 Single particle analysis of the drug loading capacity of SEAL‐prepared Dox‐mEVs by nFCM. (a) Representative SS and FL‐Dox burst traces of the Dox‐mEVs prepared by passive incubation (i, ii) and SEAL (iii, iv). (b) Bivariate dot plots of SS verse FL‐Dox of SEAL‐prepared Dox‐mEVs. (c) SS (i) and FL‐Dox (ii) intensity distribution histograms of the two subpopulations in SEAL‐prepared Dox‐mEVs. (d) Particle concentration and the active loading fraction of Dox‐mEVs prepared and purified by various methods. Note. The numbers on the above of each column indicated the active loading fractions. (e) The median SS (i) and FL‐Dox (ii) intensity of Dox‐mEVs prepared and purified by various methodsCC BY-NC · PubMed Central
FIGURE 3 Multi‐parameter analysis of the correlation between drug loading capacity and lipid labelling pattern of Dox‐mEVs. (a) Representative SS, FL‐Dox, and FL‐DiD burst traces of Dox‐mEVs without (i, ii, iii) or with (iv, v, vi) DiD labelling. (b) Bivariate dot plots of SS verse FL‐DiD. (c) Bivariate dot plots of FL‐DiD verse FL‐Dox. (d) A Venn diagram showing the overlap of different subpopulations in SEAL‐prepared Dox‐mEVs. (e) Schematic illustration of the lipid‐probe based magnetic purification process. (f) Bivariate dot‐plots of SS versus FL‐Dox and the corresponding SS and FL‐Dox histograms of the LPMIT‐purified Dox‐mEV samples
FIGURE 3 Multi‐parameter analysis of the correlation between drug loading capacity and lipid labelling pattern of Dox‐mEVs. (a) Representative SS, FL‐Dox, and FL‐DiD burst traces of Dox‐mEVs without (i, ii, iii) or with (iv, v, vi) DiD labelling. (b) Bivariate dot plots of SS verse FL‐DiD. (c) Bivariate dot plots of FL‐DiD verse FL‐Dox. (d) A Venn diagram showing the overlap of different subpopulations in SEAL‐prepared Dox‐mEVs. (e) Schematic illustration of the lipid‐probe based magnetic purification process. (f) Bivariate dot‐plots of SS versus FL‐Dox and the corresponding SS and FL‐Dox histograms of the LPMIT‐purified Dox‐mEV samplesCC BY-NC · PubMed Central
FIGURE 4 Correlation analysis between drug loading capacity and the immunofluorescence against casein protein of LPMIT‐purified Dox‐mEVs. (a) Bivariate dot plots of SS verse FL‐Cas. (b) Bivariate dot plots of FL‐Dox verse FL‐Cas. (c) A Venn diagram displaying the overlap of different subpopulations. (d) Schematic illustration of the IMIT process for the removal of casein assemblies. (e) Bivariate dot‐plots of SS versus FL‐Dox and the corresponding histograms of the purified Dox‐mEV samples
FIGURE 4 Correlation analysis between drug loading capacity and the immunofluorescence against casein protein of LPMIT‐purified Dox‐mEVs. (a) Bivariate dot plots of SS verse FL‐Cas. (b) Bivariate dot plots of FL‐Dox verse FL‐Cas. (c) A Venn diagram displaying the overlap of different subpopulations. (d) Schematic illustration of the IMIT process for the removal of casein assemblies. (e) Bivariate dot‐plots of SS versus FL‐Dox and the corresponding histograms of the purified Dox‐mEV samplesCC BY-NC · PubMed Central
FIGURE 5 Analysis of the cell cytotoxicity and cell internalization ability of cargo‐loaded mEVs. (a) CCK‐8 based viability measurement of HepG2 cells incubated with blank mEVs, doxorubicin encapsulated liposomes (Dox‐LPs), Dox‐mEVs (purified) and free doxorubicin at various concentration ranging from 0.1 to 10 μM, respectively (n = 3, mean ± SD). Note: the particle concentration of blank mEVs was identical to Dox‐mEVs. (b) CCK‐8 based viability measurement of HepG2 cells incubated with mEVs and the Dox‐mEVs prepared by various methods. p‐Values were calculated by t‐test. **p < 0.01, ***p < 0.001 (n = 3, mean ± SD). (c) Confocal fluorescence microscopy analysis of the cellular internalization of AF647‐labeled AO‐mEVs (i, AO‐mEVs‐AF647), as well as the transferrin conjugated counterpart (AO‐mEVs‐AF647‐Tf) without (ii) and with the two‐step magnetic purification (iii). Note: HepG2 cells were stained with Hoechst 33342, and excited with 405, 488, and 638 nm lasers. Scale bars are 20 μm. (d, e) FCM analysis of the fluorescence intensity of AO (d) and AF647 (e) of HepG2 cells incubated with mEVs, AO‐mEVs‐AF647, AO‐mEVs‐AF647‐Tf without and with purification, respectively
FIGURE 5 Analysis of the cell cytotoxicity and cell internalization ability of cargo‐loaded mEVs. (a) CCK‐8 based viability measurement of HepG2 cells incubated with blank mEVs, doxorubicin encapsulated liposomes (Dox‐LPs), Dox‐mEVs (purified) and free doxorubicin at various concentration ranging from 0.1 to 10 μM, respectively (n = 3, mean ± SD). Note: the particle concentration of blank mEVs was identical to Dox‐mEVs. (b) CCK‐8 based viability measurement of HepG2 cells incubated with mEVs and the Dox‐mEVs prepared by various methods. p‐Values were calculated by t‐test. **p < 0.01, ***p < 0.001 (n = 3, mean ± SD). (c) Confocal fluorescence microscopy analysis of the cellular internalization of AF647‐labeled AO‐mEVs (i, AO‐mEVs‐AF647), as well as the transferrin conjugated counterpart (AO‐mEVs‐AF647‐Tf) without (ii) and with the two‐step magnetic purification (iii). Note: HepG2 cells were stained with Hoechst 33342, and excited with 405, 488, and 638 nm lasers. Scale bars are 20 μm. (d, e) FCM analysis of the fluorescence intensity of AO (d) and AF647 (e) of HepG2 cells incubated with mEVs, AO‐mEVs‐AF647, AO‐mEVs‐AF647‐Tf without and with purification, respectivelyCC BY-NC · PubMed Central

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Metadata

Authors
Chen, C., Sun, M., Wang, J., Su, L., Lin, J., & Yan, X.
Venue
Journal of Extracellular Vesicles
Publisher
Wiley
Published
2021-10-31
Volume
10
Issue
13
ISSN
2001-3078
DOI
10.1002/jev2.12163
PMC
PMC8558234
Cited by
163
References
6
Language
en
Licence
creativecommons.org/licenses/by-nc/4.0/ (vor)
Funders
  • Natural Science Foundation of Fujian Province (2019J05097)
  • National Natural Science Foundation of China (21627811, 21934004)

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