Clinical Pharmacology & Biopharmaceutics
Open Access

Exosomes; Drug delivery; Precision medicine; Drug encapsulation; Release profiles; Engineering exosomes; Surface modification; Controlled release; Targeted therapy; Bioactive molecules; Extracellular vesicles; Therapeutic efficacy

Introduction

The field of precision medicine is rapidly advancing, focusing on tailoring medical treatments to individual characteristics, including genetic profiles, lifestyle, and environmental factors. Central to this approach is the development of more efficient drug delivery systems that can target specific cells or tissues with minimal side effects. Traditional drug delivery methods, such as oral medications or systemic injections, often face challenges related to poor bioavailability, non-specific distribution, and lack of controlled release. These limitations necessitate the exploration of alternative, more sophisticated drug delivery systems [1].

Exosomes, naturally occurring extracellular vesicles (EVs) secreted by various cells, have emerged as promising candidates for precision medicine applications. These small, membrane-bound vesicles are capable of carrying a diverse range of bioactive molecules, including proteins, lipids, and nucleic acids, offering a versatile platform for drug encapsulation. Moreover, exosomes are inherently biocompatible, non-immunogenic, and capable of crossing biological barriers, such as the blood-brain barrier, which makes them ideal for delivering therapeutic agents in various diseases, including cancer, neurodegenerative disorders, and autoimmune diseases.

However, several challenges still need to be addressed to unlock the full potential of exosomes for drug delivery. One of the primary obstacles is the optimization of exosome drug encapsulation efficiency and the control over their release profiles. In their natural state, exosomes often have low drug-loading capacity, and their release rates can be unpredictable. Thus, engineering exosomes to improve these characteristics is critical for their clinical translation.

Recent advances in exosome engineering have introduced several strategies to enhance drug encapsulation and release properties. These strategies include surface modifications, genetic engineering, and incorporation of synthetic materials into exosomes. Surface modification can improve exosome stability, target specificity, and cellular uptake, enabling precise delivery to the intended site of action. Genetic engineering allows for the manipulation of exosome cargo, ensuring efficient encapsulation of therapeutic agents and enhancing the release control. Additionally, the incorporation of synthetic lipids or polymers can help stabilize exosomes and modulate their drug release kinetics [2].

In this context, understanding the mechanisms behind exosome formation, cargo loading, and release is essential for developing more efficient drug delivery systems. By harnessing these engineering techniques, it is possible to design exosome-based systems with tailored release profiles that can precisely control the timing and dose of drug delivery, improving therapeutic efficacy and minimizing side effects. These engineered exosomes hold great promise for advancing precision medicine, offering personalized, effective treatments that can target the root causes of diseases while enhancing the quality of patient care.

This review will explore the novel approaches being developed to improve the drug encapsulation and release profiles of exosomes, highlighting key engineering strategies, current challenges, and future directions for the clinical use of exosome-based drug delivery systems. Through these innovations, exosomes may become a cornerstone in the pursuit of more effective and targeted therapies for a wide array of diseases [3].

Materials and methods

The engineering of exosomes for improved drug encapsulation and release profiles involves several key steps, including the isolation and characterization of exosomes, modification of their surfaces, and loading of therapeutic agents. In this section, we outline the materials and experimental methods used to engineer exosomes for precision medicine applications, including the strategies for improving drug encapsulation and controlling release.

Exosome isolation

Exosomes were isolated from conditioned media of cultured mammalian cells, such as human mesenchymal stem cells (hMSCs) or HEK293 cells, using differential centrifugation, ultracentrifugation, or commercial exosome isolation kits.

Materials

Cell culture medium (e.g., DMEM, RPMI-1640)

Cell lines (e.g., human mesenchymal stem cells, HEK293 cells)

Ultracentrifuge (Beckman Coulter, SW 41 Ti rotor)

Exosome isolation kits (e.g., Total Exosome Isolation Kit from Invitrogen) [4].

Method

Cells were cultured in appropriate media under standard conditions (37°C, 5% CO₂).

After 48-72 hours of incubation, conditioned media were collected, and cell debris was removed by centrifugation at 300 × g for 10 minutes.

The supernatant was subjected to ultracentrifugation at 100,000 × g for 2 hours to isolate exosomes. The pellet was resuspended in phosphate-buffered saline (PBS) for further analysis or modification.

Characterization of exosomes

Exosomes were characterized to confirm their size, morphology, and surface markers using several analytical techniques, including nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blot analysis for exosomal markers [5].

Materials

Nanoparticle tracking analysis (NTA) system (e.g., Malvern NanoSight)

Transmission electron microscope (TEM, JEOL JEM-1400)

Western blot reagents (e.g., anti-CD63, anti-CD9 antibodies)

Bradford protein assay kit (Bio-Rad)

Method

Size and concentration: Exosome size and concentration were determined using NTA. The exosome suspension was diluted and analyzed using the NTA system to measure the particle size distribution.

Morphology: Exosome morphology was examined by negative staining and imaging using TEM.

Exosomal markers: Western blotting was used to confirm the presence of exosome-specific surface markers, such as CD63, CD9, and TSG101. [6].

Surface modification of exosomes

To improve the drug delivery potential of exosomes, surface modification strategies, such as lipid bilayer modification and the conjugation of targeting ligands, were employed. These modifications aimed to enhance exosome stability, targeting specificity, and cellular uptake.

Materials

Lipid-polymer conjugates (e.g., PEGylated lipids, lipids containing targeting peptides)

Targeting peptides or antibodies (e.g., HER2 targeting peptides)

Electroporation system (e.g., BTX ECM 830) [7].

Method

Exosomes were modified by incubating them with lipid-polymer conjugates or targeting ligands.

Electroporation was used to facilitate the incorporation of these surface-modifying agents into the exosome membrane.

Surface modifications were confirmed using fluorescence microscopy and flow cytometry.

Drug loading into exosomes

Therapeutic agents, such as chemotherapeutic drugs, small molecules, or RNA-based therapeutics, were encapsulated within exosomes using various techniques, including passive loading, sonication, or electroporation.

Materials

Drug or therapeutic cargo (e.g., doxorubicin, siRNA, mRNA)

PBS (Phosphate-buffered saline)

Sonicator or electroporation system (e.g., Fisher Scientific, BTX ECM 830)

Ultracentrifuge for drug-loaded exosome separation

Method

Passive loading: Exosomes were incubated with the drug in PBS at room temperature or 4°C for several hours to allow the passive encapsulation of the therapeutic cargo.

Sonication or electroporation: For more efficient loading, the drug was introduced into exosomes using sonication or electroporation. The drug-loaded exosomes were separated from unencapsulated drug by ultracentrifugation.

The drug loading efficiency was determined by measuring the concentration of the drug in the exosome pellet using high-performance liquid chromatography (HPLC) or fluorescence spectroscopy [8].

Release studies of encapsulated drugs

The release profiles of drugs encapsulated in exosomes were assessed in vitro under different conditions, such as physiological pH and temperature, to simulate drug release in vivo. Release kinetics were studied over time to understand the control over drug release.

Materials

Release buffer (e.g., PBS, pH 7.4, pH 5.5 for acidic conditions)

Dialysis membranes (e.g., MWCO 10,000 Da)

HPLC or fluorescence spectroscopy for drug quantification

Method

Drug-loaded exosomes were incubated in release buffers at 37°C or 4°C to mimic in vivo conditions.

At predetermined time intervals, the release buffer was collected, and the amount of released drug was quantified usIng HPLC or fluorescence spectroscopy.

The release profile was plotted, and kinetic models (e.g., zero-order, first-order, Higuchi model) were used to evaluate the release mechanism [9].

Cellular uptake and cytotoxicity assays

The cellular uptake of exosomes and their therapeutic efficacy were assessed in vitro using appropriate cell lines, such as cancer cells or neural cells. Cytotoxicity assays (e.g., MTT or cell viability assays) were used to evaluate the therapeutic effects of drug-loaded exosomes.

Materials

Cancer cell lines (e.g., MCF-7, HeLa cells)

Flow cytometer for exosome uptake analysis

MTT or cell viability assay kits (e.g., Promega)

Fluorescently labeled exosomes (e.g., exosomes labeled with PKH67 dye)

Method

Cells were seeded in 96-well plates and incubated with drug-loaded or surface-modified exosomes for a specified time.

Cellular uptake of exosomes was analyzed by flow cytometry or fluorescence microscopy.

Cytotoxicity was measured using MTT or other viability assays to assess the therapeutic potential of the drug-loaded exosomes [10].

Statistical analysis

Data were expressed as the mean ± standard deviation (SD). Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. A p-value of less than 0.05 was considered statistically significant.

Discussion

Exosome-based drug delivery systems have garnered significant attention in the field of precision medicine due to their inherent biocompatibility, ability to cross biological barriers, and potential for targeted drug delivery. However, for exosomes to be effective therapeutic vehicles, significant challenges must be addressed, particularly regarding drug encapsulation efficiency, controlled release profiles, and targeted delivery. Engineering exosomes to overcome these limitations is crucial for their successful clinical application.

One of the most pressing challenges is optimizing the drug encapsulation process. While exosomes can naturally carry a variety of cargo, their low drug-loading capacity often limits their therapeutic potential. Several strategies have been developed to improve encapsulation efficiency, including the use of electroporation, sonication, and lipid-polymer conjugates. These techniques enable more efficient loading of therapeutic agents, such as small molecules, RNA-based therapeutics, and proteins, into exosomes. However, optimization is still needed to prevent the leakage of the loaded cargo and ensure stable retention during circulation in the body.

Surface modification plays a pivotal role in enhancing exosome functionality. Modifying exosomal membranes with targeting ligands, such as antibodies or peptides, can significantly improve the specificity of drug delivery. This allows for selective targeting of diseased cells, such as cancer or neuronal cells, while minimizing off-target effects. Additionally, surface modifications can increase the stability of exosomes and reduce their clearance by the immune system, thus prolonging circulation time. The incorporation of polyethylene glycol (PEG) or other stabilizing agents has proven effective in enhancing the pharmacokinetics of exosomes, ensuring that they can accumulate at the site of action.

Drug release profiles are another critical consideration for exosome engineering. To achieve controlled and sustained drug release, researchers have explored methods such as adjusting the lipid composition of exosomal membranes or incorporating responsive materials that release drugs in response to specific stimuli (e.g., pH, temperature, or enzyme activity). By fine-tuning these parameters, it is possible to create exosome-based systems that release their payload at the optimal time and location, thereby improving therapeutic outcomes.

Recent advances in genetic engineering have also paved the way for more sophisticated exosome-based delivery systems. The ability to modify the cargo carried by exosomes through genetic manipulation opens new possibilities for precision medicine. For instance, RNA-based therapies, such as siRNA or mRNA, can be efficiently loaded into engineered exosomes for gene silencing or protein replacement therapies. These advancements hold great promise for treating genetic disorders, cancer, and other diseases with high unmet medical needs.

Despite the tremendous potential of exosome-based drug delivery systems, several challenges remain before they can be widely adopted in clinical practice. Standardization of exosome isolation and characterization techniques is essential for ensuring reproducibility and quality control. Additionally, scaling up production while maintaining exosome integrity and functionality is a significant hurdle that requires further development.

Conclusion

The engineering of exosomes for precision medicine represents a promising frontier in drug delivery systems, offering a versatile, biocompatible platform for the targeted treatment of diseases. Exosomes, due to their natural origin and ability to transport a variety of bioactive molecules, present unique advantages over traditional drug delivery methods. However, to fully realize their therapeutic potential, challenges related to drug encapsulation efficiency, controlled release, and targeting specificity must be overcome.

Recent advancements in exosome engineering have significantly improved drug encapsulation and release profiles, using methods such as electroporation, sonication, and lipid-polymer conjugates. These innovations have enhanced the loading capacity of exosomes, enabling the efficient delivery of a wide range of therapeutic agents, from small molecules to RNA-based therapeutics. Surface modifications, including the incorporation of targeting ligands, have further increased the specificity of exosome-mediated drug delivery, ensuring that therapeutic payloads are directed to the intended cells or tissues, minimizing off-target effects, and improving therapeutic outcomes.

The ability to control drug release is another critical advancement in exosome engineering. Through adjustments to exosome composition, such as lipid modifications or the use of responsive materials, researchers can tailor release profiles to match specific therapeutic needs. This capability allows for the controlled and sustained delivery of drugs, optimizing therapeutic effects while minimizing side effects. Furthermore, genetic engineering of exosomes opens up new possibilities for personalized medicine by enabling the design of exosome-based systems that carry and release specific RNA molecules for gene therapies.

Despite the tremendous progress, challenges remain in the large-scale production and clinical translation of engineered exosomes. Standardization of isolation and characterization protocols, alongside improvements in scalability, are necessary to ensure reproducible and efficient production of exosome-based therapeutics. Additionally, long-term studies are required to assess the safety, immunogenicity, and efficacy of engineered exosomes in clinical settings.

In summary, engineered exosomes have the potential to revolutionize the field of precision medicine by providing a highly adaptable and targeted drug delivery platform. Continued research into improving encapsulation efficiency, controlling release kinetics, and enhancing targeting capabilities will be crucial for advancing exosome-based therapies. As these technologies evolve, exosome-based systems may offer more personalized, effective, and safer treatment options for a wide range of diseases, including cancer, neurological disorders, and genetic diseases, ushering in a new era of medicine tailored to individual patient needs.

Conflict of interest

None

Acknowledgment

None

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