Innovative Drug Delivery Systems for Precision Medicine

The definition of “drugs” is expanding with advances in biotechnology. Researchers have developed a variety of drugs to treat diseases, ranging from small molecules to proteins and nucleic acids, such as DNA, small interfering RNA (siRNA) and messenger RNA (mRNA). The success of mRNA vaccines during the COVID-19 pandemic and recent progress in gene therapies have highlighted the potential of molecular cargo as medicine.

However, one major hurdle for precision medicine is getting the molecular cargo to its desired tissues and cells. Therapeutic cargo like proteins and nucleic acids can already be manufactured at scale, but if they are not delivered to the target tissues, efficacy will be low. When these drugs accumulate in non-target tissues in large quantities, it can also lead to safety concerns. This article explores emerging technologies using materials such as lipid nanoparticles and membrane-coated nanovesicles to enhance the precision, efficacy and safety of drug delivery.

DNA barcoding for high-throughput in vivo screening

When drug delivery agents such as lipid nanoparticles are introduced into the body, it is challenging to control where they end up. This is because in vivo biodistribution is affected by multiple factors, such as circulation and tissue barriers, in addition to the presence of proteases that can degrade the drug delivery materials.

Typically, to develop a tissue- or cell-targeting drug delivery vehicle, researchers would first need to test a library of materials using in vitro models such as cells, spheroids and organoids. However, it is well known that in vitro models do not reflect the complexity of in vivo conditions. On the other hand, it will be resource-intensive to test all delivery vehicles in animals.

Barcoding is a very useful approach to enable high-throughput in vivo screening. Imagine when someone goes to a supermarket and does a self-checkout; the machine will know what items they want to buy simply by scanning the printed barcodes.

Optimize Nanomedicine With Better Particle Insight

Explore the most effective techniques for analyzing nanoparticle properties
View Infographic

Advertisement

With this same concept, unique DNA barcodes can be tagged on each species of drug delivery vehicle, and the vehicles can be administered all at once into the animals. Depending on the tissue or organ of interest, after delivery, the tissue can be removed, digested and sequenced for the presence of DNA barcodes. If DNA barcode A is detected at the highest frequency, it would mean that the drug delivery vehicle that was tagged with DNA barcode A accumulated the most in the target tissue. This approach enables many drug delivery vehicles to be screened for their in vivo biodistribution all at once, greatly reducing the time and costs associated with animal studies.

Mukalel and colleagues made use of this approach to screen and identify an oxidized lipid nanoparticle that transfects monocytes for eliminating B cells expressing CD19.¹ Eliminating B cells can be useful for diseases such as B-cell leukemia and autoimmune diseases due to abnormal antibody secretions. Challenges associated with chimeric antigen receptor (CAR) T-cell therapy, such as poor tumor infiltration and toxicity, may be addressed with CAR-monocyte and CAR-macrophage (CAR-M) therapy. However, making CAR-M ex vivo remains costly and logistically inconvenient. Mukalel and team are motivated to develop in situ CAR-M manufacturing as it will make this therapy more cost-effective and accessible to more patients. However, there is still concern over the specificity of cell targeting.

Mukalel et al. first tapped into the affinity of myeloid cells for oxidized lipids to design a library of ionizable lipid structures with different degrees of oxidation. The ionizable lipids were then combined with other ingredients and mixed with an aqueous phase containing luciferase mRNA via a microfluidic device to form lipid nanoparticles. The mRNA serves as a biomarker to demonstrate that the cargo has been delivered intact and can be translated into visual protein signals. The additional ingredients serve to promote endosomal escape, stabilize lipid nanoparticles and enhance the circulation time of the nanoparticles.

Next, the team tested their materials on human monocyte cell lines (THP-1) and primary macrophages and found that the oxidized lipid nanoparticles always outperformed their non-oxidized counterparts. Interestingly, they found that ionization could affect the escape of lipid nanoparticles from endosomes, which was beneficial for transfecting highly phagocytic cells like macrophages. This is because the oxidized materials enabled cargo to escape from the acidic subcellular compartments in macrophages more efficiently.

When the team moved on to in vivo screening, they found that the top-performing lipid nanoparticles included both oxidized and unoxidized vehicles, suggesting the complexity of in vivo tissue- and cell-targeting for precision medicine. The authors argued that factors such as the role of the binding of serum proteins, formation of a protein corona and biotransformation by the liver could also affect the tissue tropism and stability of drug delivery vehicles. They emphasized why it is crucial to move beyond using in vitro models only for nanoparticle testing. Using flow cytometry, the authors discovered that phagocytic myeloid cells such as monocytes, macrophages and dendritic cells preferentially uptake the lipid nanoparticles in blood, spleen and lymph nodes.

Finally, using their top performer, C14-O2, the authors delivered mRNA encoding for anti-CD19 CAR. Anti-CD19 CAR was detected in 2% of circulating monocytes but not in CD3+ T cells or CD19+ B cells. This treatment also reduced the percentage of B cells by 45% compared to control mice.

These results show how chemical composition can impact delivery performance. Beyond chemistry, however, physical properties like particle shape also play an important role.

The effects of shape on nanoparticle uptake

A tumor contains multiple cell types, including cancer cells, endothelial cells and immune cells. Research has found that each cell type may have a preference for different nanoparticle shapes, but this has thus far been shown in vitro and not in animals.

Huang and colleagues applied the DNA barcoding approach to tag six gold nanoparticles (two sizes and three shapes) with unique DNA barcodes.² However, as the barcoding approach has not been used for inorganic drug delivery vehicles, they conducted a series of tests to show that the barcode types did not interfere with cell uptake, the barcodes had minimal detachment after vigorous shaking and the barcodes were resistant towards DNAse degradation. Using this system, they found that 4T1 breast cancer cells preferred prism-shaped gold nanoparticles to rod-shaped gold nanoparticles, and the former was able to deliver siRNA with better efficacy. Nonetheless, it is not trivial to synthesize nanoparticles of different shapes. For instance, most lipid-based nanocarriers are based on spherical shapes, but an increasing amount of research highlights that shape can influence in vivo biodistribution, tissue tropism and cell targeting.³ Currently, the assembly of lipid-based nanoparticles into non-spherical morphologies requires the addition of synthetic polymers, proteins or lipid-polymer conjugates, which introduce challenges like higher immunogenicity and complement activation.

To overcome this limitation, Pires and colleagues introduced charged lipid headgroups to generate stable discoidal lipid nanoparticles from mixed micelles.⁴ They hypothesized that lipids containing two charges in proximity could increase the lipid headgroup charge density and enable nanodiscs to remain stable in physiological ionic strength buffers without the need for lower ionic strength buffers, which only produced very small unilamellar liposomes that were unstable. By measuring the size and zeta potential of the new materials, the authors confirmed that stable nanodiscs could be formed with their method.

Next, the authors coated their nanodiscs with a thin bilayer of poly-L-arginine and poly-L-glutamate, which they had previously found to promote binding of the nanoparticles to the surface of ovarian cancer cells without triggering endocytosis. Mice given the bilayer-coated nanodiscs had greater accumulation of the nanocarriers in the tumor, supporting that the nanodisc morphology with an ovarian cancer-targeting bilayer coating substantially enhances the delivery of lipid nanoparticles to metastatic ovarian cancer. This paper demonstrates a facile approach to creating non-spherical lipid nanoparticles to improve tissue targeting for cancer therapy.

Engineered biomimetic nanovesicles for targeted drug delivery

Another promising strategy is to engineer biomimetic nanovesicles. This involves disguising nanoparticles by coating them with the membranes of desired cells, enabling them to inherit properties of the donor cells, such as immune evasion and tissue homing.

AI-Enabled Imaging Solutions for Complex 3D Organoid Screening

This application note demonstrates how next-generation confocal imaging technology combined with AI-powered analysis tools enables researchers to capture high-quality images from 3D organoids.
View App Note / Case Study

Advertisement

Ma and colleagues adopted this approach to overcome the challenges associated with delivery across the bone marrow-blood barrier. They isolated cell membrane from bone marrow stromal cells (BMSCs) as this cell type has a natural ability to infiltrate the marrow vasculature and home to the bone marrow.⁵ The membrane was used to coat poly(lactic-co-glycolic acid) (PLGA) to deliver teriparatide (TPD) for osteoporosis, and tetrahedral framework nucleic acid (tFNA) to deliver an acid-releasing system of doxorubicin for osseous metastasis and systemic metastatic breast cancer.

To improve understanding of how membrane proteins could affect bone homing, immunogenicity, circulation time and drug loading, the authors performed a proteomic analysis of the surface proteins retained on the membrane. They found that the key markers of bone marrow stromal cells and bone-specific proteins, such as CD151 and CD276, were preserved on the biohybrid nanoparticles. Proteins, including CD47 and CD44, that are beneficial to evade immune detection were also preserved.

Using the biomimetic PLGA nanoparticles, the authors showed that delivery of TPD increased the serum level of calcium and osteocalcin, and the activity of osteoblasts was also higher. Measurement of trabecular bone mineral density further confirmed that the biomimetic nanoparticles were better able to retain bone mass to counter the effects of osteoporosis. Finally, the team created a mouse model of breast-to-bone cancer metastasis to demonstrate that the biomimetic tFNA nanoparticles were able to deliver doxorubicin to bone tissues and reduce the severity of metastasis with greater efficacy than unmodified nanocarriers.

“In vitro experiments showed that biomimetic PLGA nanoparticles delivered the most TPD to BMSCs, indicating enhanced cellular uptake attributable to the BMSC-derived membrane coating. However, the extent to which this uptake reflects the selective homologous targeting of BMSCs remains uncertain and warrants further validation through comparative studies using non-BMSC cell types. Moreover, the precise mechanisms underlying the internalization of biomimetic PLGA nanoparticles remain to be elucidated. It is still unclear whether uptake occurs primarily via membrane fusion, receptor-mediated endocytosis, or a combination of pathways,” explained Wei Seong Toh, associate professor and research director at the Yong Loo Lin School of Medicine, National University of Singapore.

Toh added that “future studies should aim to dissect these mechanisms using pathway-specific inhibitors, real-time live cell imaging and intracellular co-localization analyses to better understand the cellular trafficking dynamics and targeting efficiency of these bioengineered nanoparticles. Critically, such insights will also help determine whether biomimetic PLGA nanoparticles facilitate endosomal escape, thereby enabling the effective cytosolic delivery of TPD – an essential step for maximizing therapeutic efficacy.”

Expanding the drug delivery toolbox

Advances in biomedicine have identified an expanding library of molecular targets and their corresponding therapeutic cargo for treatments. However, precision medicine can only be achieved when the right medicine is given at the right dose, right time and right place. Drug delivery vehicles play a crucial role in enabling efficacious and safe treatment.

DNA barcoding approaches will enable us to identify materials with tissue- and cell-targeting ability, while improvements in synthesis can expand our material library for screening. Beyond synthetic materials, it is also useful to consider how biohybrids can be exploited, such as harvesting cell membranes as decoys to allow synthetic nanoparticles to escape from immune surveillance or as boosters to enhance transport to the target tissues and cells. All these advances will move the field closer to realizing precision delivery for the next generation of molecular medicines.