Drug & Gene delivery

Gene delivery vectors are engineered vehicles designed to transport therapeutic genetic material (DNA & RNA) into target cells to treat or prevent disease. They overcome the natural barriers cells have against foreign genetic material. Major vector classes include viral vectors (Biological) and Non-viral vectors (Synthetic/Chemical). The benefit of gene delivery vectors is that they are the essential engines driving gene therapy and genetic medicine. The choice between viral and non-viral systems involves a careful trade-off between efficiency, cargo capacity, duration, immunogenicity, safety, manufacturability, and cost.

Viral systems

Viral vectors exploit natural viral infection mechanisms. The advantages of these vectors consist of high efficiency of delivery, long-term expression, specific targeting, and well-studied and developed. The most common viral vectors are lentivirus, baculovirus, retrovirus, and adeno-associated virus (AAV). Most of these viral vehicles produce permanent expression, while adenoviruses create transient expression. Viral vectors are the go-to choice when long-term, high-level gene expression is required for a disease (e.g., hereditary disorders like hemophilia or SMA), and the risks of immunogenicity and mutagenesis can be managed.
Some vectors, like retrovirus, lead to the integration of the transgene into the host genome, which results in limitations in some activities, such as activation of oncogenes, insertional mutagenesis, packaging capacity of exogenous DNA, and neutralizing antibodies against AAV. To solve this problem, non-integrating vectors were designed to bypass activating oncogenes and achieve targeted integration by applying zinc finger nuclease (ZFN) to add the erythropoietin gene (Epo) into the chemokine (C-C motif) receptor-5 gene locus of cells.
Immunogenicity is another challenge in viral vectors that liver-targeted expression to deliver the gene to liver cells (hepatocytes) can promote tolerance to the transgene product. Using alternative technologies such as synthetic vectors like lipid nanoparticles (LNPs) or polymers to deliver the genetic material, mRNA COVID-19 vaccines are a famous example of LNP delivery. Gene editing, such as CRISPR-Cas9 system, instead of adding a new gene, aims to directly correct the mutation in the patient’s own DNA. Furthermore, since a large portion of the human population can neutralize AAV antibodies, the in vivo effect decreases dramatically. Hence, AAV vectors may be among the most promising vectors because of their reduced pathogenicity in humans and their ability to achieve long-term gene expression.
Packaging of viral vectors
Both AAV and lentivirus are produced using a method called transient transfection. Common procedures used for transfecting cells include chemical and physical methods. Chemical methods include polymeric carriers, lipid agents, inorganic nanoparticles, and dendrimers. Using inorganic nanoparticles (NPs) for transfecting cells is frequently associated with polycations. Lipid-Based Nanoparticles (LNPs) consist of cationic/ionizable lipids + helper lipids (phospholipids, cholesterol) + PEG-lipid encapsulating nucleic acids. Physical methods that directly perturb cell membranes include electroporation, nucleofection, gene gun, sonoporation, and hydrodynamic delivery. Choosing the transfection procedure or reagent is essential for increasing the transfection rate.
The fundamental principle is to transfer human embryonic kidney (HEK) 293 cells (or a derived cell line like 293T) with a set of plasmids that provide all the necessary components to make the virus, but are themselves unable to be packaged. The key feature of the packaging is its ability to infect both dividing and non-dividing cells by actively importing its genetic material into the nucleus. In the packaging procedure, replication-incompetent virus (safe for use) is used. For this reason, the viral genome is split across multiple plasmids to prevent the creation of a replication-competent virus.
There are three groups of plasmids in lentiviral packaging, which consist of the transfer plasmid (interest gene), packaging plasmids(Often split into two plasmids), and envelope plasmid.
The packaging procedure started by culturing HEK 293T cells (chosen for high transfection efficiency) to ~70-80% confluency. Transfection via co-transfection of the cells with the three (four) plasmids at an optimized ratio (e.g., Transfer, Packaging, and Envelope packaging 2:1:1) using different transfection reagents like PEI, calcium phosphate, and lipofectamine. Incubation of the cells to take up the plasmids and use their own machinery to express the viral proteins.
Virus assembly is done for the viral proteins and genomic RNA is assembled at the cell membrane and buds out. Harvesting step is done 48 and 72 hours post-transfection, the culture medium (containing the viral particles) is collected. Concentration & Purification is an important step where the medium is centrifuged at low speed to remove cell debris. The virus in the supernatant is then concentrated (often by ultracentrifugation) and/or purified (using chromatography or gradient centrifugation). Titration of the concentrated virus is quantified (titered) to determine its concentration (e.g., Transducing Units per mL – TU/mL) using methods like qPCR (genomic titer) or functional assays on target cells (functional titer). Storage of viral particles at -80°C.
One of the major challenges in the viral transfection procedure consists of delivery efficiency and cytotoxicity. There are some procedures for solving this problem, such as optimizing reagent-to-DNA ratios, using next-generation transfection reagents that are specifically formulated for viral production, and selecting alternative methods, including Calcium Phosphate Transfection: A classic, low-cost method that works very well for HEK 293 cells. It requires precise timing and pH control but can yield high titers with minimal cost. PEI (Polyethylenimine) Transfection: A very popular, cost-effective polymer for large-scale viral vector production. High-quality, linear PEI (e.g., PEIpro®) is an industry standard due to its good efficiency and scalability. Baculovirus/Sf9 System (for AAV): For certain vectors like AAV, using insect Sf9 cells infected with recombinant bacmams (baculoviruses carrying the necessary genes) can be more scalable and productive than mammalian cell transfection.
The choice between using lentiviruses or AAV depends on the experimental skill. For example, lentiviruses are used for integrating into difficult-to-transfect cells (like stem cells) or for long-term expression in dividing cells. AAV is used for efficient in vivo gene delivery to non-dividing cells with a superior safety profile.

Non- viral systems

Non-viral systems are preferred for applications where transient expression is sufficient (e.g., cancer therapy, vaccines, CRISPR gene editing), safety is the paramount concern, or a large gene needs to be delivered.
Advantages of non-viral vectors consist of low immunogenicity, no risk of insertional mutagenesis, large cargo capacity, ease of manufacturing and cost-effectiveness, and flexibility and design control.
Limitations of non-viral vectors include low transfection efficiency, which can be modified by incorporating cell-penetrating peptides (CPPs), targeting ligands, and nuclear targeting in the cargo to facilitate nuclear entry, transient gene expression that can be improved by utilizing CRISPR/Cas9 or transposon systems (e.g., Sleeping Beauty) for targeted genomic integration and engineered mRNA with modified nucleosides for enhanced stability and prolonged expression, cytotoxicity that can be overcomed by biodegradable polymers and natural & less toxic lipids, and difficulty with in vivo delivery that can be modified by polyethylene glycol (PEG) or other hydrophilic polymers, active targeting peptides, or antibodies, and physical methods such as electroporation, sonoporation, gene guns, and direct injection.

Non-viral transfection procedure

Transfection process is used to transfer non-viral vectors, which is achieved through chemical or physical methods. The transfection procedure has an exact protocol that varies by method and cell type, but the general workflow is consistent, which consists of cell seeding, transfection complex formation (Day of Transfection) that is the most crucial step to mix the nucleic acid with the transfection reagent to form stable, positively charged complexes called polyplexes (with polymers) or lipoplexes (with lipids), delivery to cells for adding complexes to cells, post-transfection & analysis through media changing, incubation and expression, and analysis for evaluation based on microscopy, flow cytometry, qPCR/RT-PCR, western blot or ELISA.
Some of the important challenges in the non-viral transfection procedure consist of low cellular uptake, which is reported because the negatively charged cell membrane repels the naked nucleic acid. Hence, modification of surface charge and creating vectors with a slightly positive charge (cationic) promotes interaction with the negative cell membrane. Conjugating specific molecules (e.g., antibodies, peptides, folate, carbohydrates) to the vector’s surface that bind to receptors on the target cell is another solution.
Endosomal entrapment and degradation is the biggest bottlenecks. Most of the vectors are taken up by the cell via endocytosis, becoming trapped in an endosome and degraded. So, polymers like PEI with a high buffering capacity absorb protons (H+ ions) in the acidic endosome and cause chloride ions (Cl-) to flood in. Moreover, using endosomolytic agents, such as incorporating peptides or other agents that are activated at low pH to disrupt the endosomal membrane.

Electroporation

Electroporation is a common procedure of transfection in non-viral vectors. Some of the challenges and solution are mentioned here. Low efficiency is overcome by systematically optimizing voltage, pulse length, MOI, and cell concentration. Damaging the virus can be overcome by using low-ionic-strength buffers and keeping samples cold. Low cell viability that can be overcome by optimizing pulse parameters (lower V, shorter time). Buffer incompatibility requires exchanging the buffer and using specialized electroporation buffers.
Our team has hands-on experience in addressing these pain points. We provide consulting services for selecting proper gene delivery vectors, improving the transfection procedure, training in advanced preparation techniques for gene targeting, and strategic guidance for increasing transfection rate, using the nuclease procedures, and regulatory compliance.
By tackling laboratory challenges, we help ensure gene delivery products reach their full potential in clinical and commercial use.

Liposomes

Liposomes are spherical vesicles composed of natural or synthetic phospholipids, widely recognized as one of the most reliable and clinically validated drug delivery systems. They have the unique ability to encapsulate both hydrophilic and lipophilic molecules, making them a versatile platform for pharmaceutical and biomedical applications. Their importance lies in protecting sensitive drugs, such as nucleic acids or peptides, from degradation while improving bioavailability and reducing systemic toxicity. Without proper formulation, however, liposomes may face issues such as poor encapsulation efficiency, drug leakage, or instability—directly affecting therapeutic success.

One of the greatest advantages of liposomes is their strong clinical track record; FDA-approved liposomal drugs like Doxil® demonstrate their potential for real-world impact. Surface modifications such as PEGylation or ligand attachment further enhance circulation time and allow targeted delivery. Liposomes have been successfully applied in cancer chemotherapy, antifungal treatments, vaccine development, and even cosmetic formulations.

Despite their promise, liposomes come with well-documented challenges. Shelf-life instability due to lipid oxidation and hydrolysis is common, but can be mitigated with antioxidants, controlled storage, or lyophilization using cryoprotectants. Drug leakage during storage and transport is another issue that often frustrates researchers; fine-tuning lipid composition and bilayer rigidity is essential to minimize this. In production, particle size heterogeneity often leads to unpredictable pharmacokinetics, requiring extrusion or microfluidic-based methods for tight size control. From an industrial perspective, high production costs and difficulties in scale-up are frequent barriers, particularly when moving from academic labs to GMP facilities. These can be addressed through continuous manufacturing systems, process automation, and careful raw material selection.

Several established methods are available for liposome preparation, each offering distinct advantages depending on the intended application, drug type, and scalability:
1. Thin Film Hydration (Bangham Method):
The classical and most widely used approach.
Lipids are dissolved in an organic solvent, dried as a thin film, and then hydrated with an aqueous buffer.
Produces multilamellar vesicles (MLVs) that can be downsized by sonication or extrusion.

2. Reverse-Phase Evaporation:
Lipids are dissolved in organic solvents and emulsified with an aqueous phase, followed by solvent removal.
Yields large unilamellar vesicles (LUVs) with relatively high encapsulation efficiency for hydrophilic drugs.

3. Ethanol or Ether Injection:
Lipid solution in a volatile solvent (ethanol or ether) is rapidly injected into an aqueous phase.
Results in small unilamellar vesicles (SUVs).
Simple and reproducible, but limited by solvent toxicity and removal challenges.

4. Microfluidics-Based Methods:
A modern, scalable technique where lipids in ethanol and aqueous drug solutions are mixed under controlled laminar flow.
Enables precise control over liposome size, polydispersity, and reproducibility.
Increasingly used in GMP manufacturing.

5. Extrusion and Sonication (Size Reduction):
MLVs can be processed through polycarbonate membranes (extrusion) or sonicated to achieve uniform nanosized vesicles.
Essential for reducing heterogeneity and improving pharmacokinetics.

6. Freeze–Thaw Cycling:
Repeated cycles of freezing and thawing improve encapsulation efficiency by disrupting and reforming the lipid bilayer.
Often combined with thin-film hydration.

Our team has hands-on experience in addressing each of these pain points. We provide consulting services for improving liposomal stability, training in advanced preparation techniques, and strategic guidance for scale-up and regulatory compliance. By tackling both laboratory and industrial challenges, we help ensure liposomal products reach their full potential in clinical and commercial use.

Niosomes

Niosomes are innovative vesicular carriers made from non-ionic surfactants and cholesterol, designed to encapsulate both hydrophilic and lipophilic drugs. Their importance in modern drug delivery lies in their ability to improve solubility, stability, and bioavailability while reducing systemic toxicity. However, if not optimized properly, niosomes may suffer from drug leakage, low encapsulation efficiency, or poor reproducibility, which can severely affect therapeutic outcomes.

One of the biggest advantages of niosomes is their cost-effectiveness and higher stability compared to liposomes, making them particularly attractive for companies seeking practical yet efficient nanocarrier systems. They are widely applied in transdermal delivery of anti-inflammatory drugs, cancer therapy, vaccine formulations, and cosmetics.

In practice, researchers often face real-world challenges when working with niosomes. Drug leakage during storage is common, but this can be mitigated by optimizing the surfactant-to-cholesterol ratio, incorporating stabilizers, or applying lyophilization techniques. Encapsulation efficiency, especially for hydrophilic drugs, may be disappointingly low, requiring fine-tuning of hydration methods and surfactant selection. At the scale-up stage, batch-to-batch variability is another frequent issue, where manual methods such as thin-film hydration are difficult to reproduce consistently. Transitioning to microfluidic or continuous production platforms can significantly improve uniformity and reproducibility. Finally, long-term stability can be affected by surfactant oxidation or aggregation, problems that can be addressed with antioxidant incorporation and optimized storage conditions.

Our consulting team specializes in troubleshooting exactly these types of problems. We guide clients in designing robust formulations, adopting industrially relevant production methods, and training staff on critical quality control steps. With the right strategies, niosomes can become a reliable and scalable solution for advanced drug delivery applications.
Several approaches are commonly used to prepare niosomes, each with specific advantages depending on the type of drug and the desired vesicle characteristics:
1. Thin Film Hydration (Conventional Method):
Non-ionic surfactants and cholesterol are dissolved in an organic solvent, which is then evaporated to form a thin lipid film.
Hydration with an aqueous buffer results in multilamellar vesicles (MLVs), which can be further downsized by extrusion or sonication.

2. Reverse Phase Evaporation:
Surfactants dissolved in organic solvents are emulsified with an aqueous phase followed by solvent removal.
Produces large unilamellar vesicles (LUVs) with high encapsulation efficiency for hydrophilic drugs.

3. Ether or Ethanol Injection:
A solution of surfactants in a volatile organic solvent (ether/ethanol) is rapidly injected into an aqueous solution.
Solvent evaporation leads to the spontaneous formation of niosomes.

4. Microfluidic Mixing:
Modern technique enabling precise control over vesicle size, uniformity, and reproducibility.
Highly scalable and increasingly used in industrial applications.

5. Sonication and Extrusion:
Applied to reduce particle size and polydispersity, leading to nanosized niosomes with improved stability.

Lipid Nanoparticles (LNPs)

Lipid nanoparticles (LNPs) represent the most advanced generation of lipid-based carriers, composed of ionizable lipids, cholesterol, phospholipids, and PEG-lipids. Their importance cannot be overstated: LNPs enabled the delivery of mRNA vaccines during the COVID-19 pandemic and are now revolutionizing gene therapy, siRNA delivery, and cancer immunotherapy. Unlike conventional carriers, LNPs are specifically engineered to encapsulate and protect fragile nucleic acids, ensuring that they reach target cells effectively. Without careful design, however, nucleic acids degrade rapidly, lose activity, or trigger unwanted immune responses.

The main advantage of LNPs is their high encapsulation efficiency and proven clinical success. They offer a safer alternative to viral vectors, with the flexibility to adjust lipid composition for tissue-specific targeting. Beyond vaccines, LNPs are opening doors for personalized medicine and rare disease therapies, where precision delivery is critical.

Yet, researchers and manufacturers often encounter serious challenges with LNPs. A major problem is stability and cold-chain dependency, as many formulations require ultra-low temperatures for storage, which complicates logistics and increases costs. This can be improved by engineering more stable lipid structures or developing lyophilized formulations. Batch-to-batch variability and reproducibility during lab preparation is another common obstacle, especially when using manual mixing methods. Moving to microfluidic systems and automated GMP-compliant platforms significantly enhances consistency.
Additionally, off-target delivery and unwanted immune activation remain major concerns, which can be addressed through careful optimization of ionizable lipids, PEGylation strategies, and surface modifications. From an industrial perspective, scaling up production while maintaining encapsulation efficiency is a recurring challenge; transitioning from bench-scale mixers to industrial continuous systems is often essential for commercialization.
Lipid nanoparticles are typically composed of ionizable or cationic lipids, helper phospholipids, cholesterol, and PEG-lipids. Their preparation methods are designed to ensure efficient encapsulation of nucleic acids (e.g., siRNA, mRNA) or small molecules while maintaining particle stability and uniformity.
1. Microfluidic Mixing (Gold Standard):
The most widely used method for clinical and industrial applications.

Lipids dissolved in ethanol are rapidly mixed with an aqueous solution of nucleic acids under controlled microfluidic flow.
Results in uniform nanoparticles (50–100 nm) with high encapsulation efficiency.
Scalable and highly reproducible, making it the preferred technique for GMP manufacturing.

2. Ethanol Injection:
Lipid solution in ethanol is injected into an aqueous phase containing the therapeutic payload.
Rapid self-assembly of lipids into nanoparticles occurs.
Simple and cost-effective but offers less control over particle size and polydispersity compared to microfluidics.

3. T-Mixer or Flash Nanoprecipitation:
A variant of controlled mixing using turbulent flow or staggered herringbone micromixers.
Allows precise particle size tuning and is adaptable to continuous manufacturing.

4. High-Pressure Homogenization:
Lipids and payload are pre-emulsified and then homogenized at high pressure.
Produces nanosized lipid particles with relatively uniform distribution.
More commonly applied to solid lipid nanoparticles but can be adapted for LNPs.

5. Solvent Evaporation or Dialysis:
Lipids dissolved in organic solvents are mixed with the aqueous phase and then subjected to solvent removal (by evaporation or dialysis).
Less common for industrial production due to low encapsulation efficiency and scalability challenges.
Our consulting and training programs are designed to help companies overcome these exact bottlenecks. We support clients in formulation optimization, troubleshooting encapsulation efficiency, improving stability for real-world conditions, and preparing scalable manufacturing strategies. By combining scientific expertise with practical solutions, we enable organizations to accelerate the development of LNP-based therapeutics from lab to market.