Nanostructured Lipid Carrier
Nanostructured Lipid Carriers (NLCs) are a type of nanoparticle drug delivery system, composed of an organic and aqueous phase in a ratio ranging from 70:30 to 99.9:0.1 (Khan, 2022), which allows accommodation for more drug molecules, compared to solid lipid nanoparticles (SLNs) (Khan, 2022). The incorporation of liquid lipids disrupts the organized crystalline structure of solid lipids, leading to an arrangement with more surface area to encapsulate drug molecules.
NLCs offer several advantages that make it a promising drug delivery system, such as a higher drug loading capacity, improved and enhanced long-term stability, longer circulation time and reducing issues such as toxicity, endosomal escape cavity, and drug expulsion. Various accessible methods are used to prepare NLCs, such as high pressure homogenization, microemulsion. However, NLC’s large molecular weight and high buffering capacity could result in poor biocompatibility and cytotoxicity. These issues can be fixed by ligand modifications and combining PEG(polyethylene glycol) with PEI (Polyethylenimine). Similar studies have modified PEI with the R18 peptide to boost encapsulation efficiency for targeted drug deliveries in dermal applications (Zheng, 2019).
In CAR_Ma, we are utilizing the NLC to package our system. Incorporating NLCs into CAR_Ma represents a significant advancement in drug delivery, as this is the first ever cancer therapeutic to be packaged into a nanoparticle system. The utilization of Nanostructured Lipid Carriers in CAR_Ma signifies a transformative approach to drug systems– by harnessing the unique structural and encapsulation properties of NLC, this innovative technology is able to make cancer therapeutics accessible and affordable, paving the way for more effective therapies and the potential of nanoparticle technology in modern cancer medicine.
NLC has also been utilized in various medical and cosmetic applications, displaying their versatility and effectiveness in the biotechnology field. In dermal drug delivery, NLC is able to increase the penetration of drugs like diclofenac and ketoprofen through the skin, which is ideal for alleviating dermatological disorders such as psoriasis and arthritis (Pardeike, 2009). In the cosmetics industry, NLCs are employed to improve the stability of active ingredients in sunscreens and moisturizers, encapsulating coenzyme Q10, which prolongs the release on skin (Ahmad, 2021). Currently, NLCs are also being explored in vaccine development. They have been successfully formulated with ribonucleic acids for Zika virus vaccines. These few applications of NLCs highlight their potential in enhancing vaccine stability and efficacy.
Zein Nanoparticle
Zein Nanoparticles (ZNPs) are storage proteins primarily obtained from corn (Zea mays). Zein is recognized for its biocompatibility, biodegradability, and GRAS (generally recognized as safe) status, which is the reason why we selected it as a material for nanoparticle drug delivery. ZNPs are particularly effective for delivering hydrophobic drugs due to their unique amphiphilic nature, able to encapsulate a myriad of drug molecules, which enhances their bioavailability. In addition, studies have demonstrated that ZNPs are able to improve the oral bioavailability of non-soluble drugs by protecting them from degradation in the gastrointestinal tract (Zhang et al, 2020).
Various methods have been developed for ZNP preparation, such as solvent evaporation, emulsification, and high shear homogenization. In the development of CAR_Ma, we leveraged Zein Nanoparticles for the encapsulation of our srRNA. The integration of this is the first in cancer therapeutics delivery, and could potentially be the most biodegradable encapsulation that exists. This innovative approach demonstrates awareness for environmentally friendly solutions to modern nanoparticle drug delivery, and opens a pathway to future protein nanoparticle technology. The application of Zein Nanoparticles in this context highlights its advantage over traditional lipid nanoparticle delivery systems, such as enhanced bioavailability. This capability is crucial in addressing the challenges associated with conventional cancer CAR therapies, which are expensive, poorly soluble, and highly toxic.
Zein Nanoparticles are extremely versatile in their other applications. For example, integrating chemotherapeutic agents with RNA interference (RNAi) technology has demonstrated potential in surmounting multidrug resistance in cancer cells (Chidambaram et al., 2011). Zein Nanoparticles are also used for encapsulating bioactive compounds such as vitamins and antioxidants. Studies have shown that using ZNPs improves stability compared to curcumin (Khan, 2020). This application extends ZNPs to food technology, enhancing the delivery of biochemicals in food products.
DOTAP
DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) is a cationic lipid that is widely used in the development of lipid nanoparticles (LNPs) for the delivery of nucleic acids such as plasmid DNA (pDNA). It is a quaternary ammonium lipid with a positive charge, and can easily bind with negatively charged nucleic acids, facilitating their encapsulation within LNPs. DOTAP has been extensively studied for its ability to deliver pDNA and antisense oligonucleotides to cells in vitro and in vivo.
DOTAP is used for many applications in drug delivery. For mRNA delivery, researchers have developed an ideal lipid nanoparticle formula composed of DOTAP, DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and cholesterol for effective mRNA delivery. This formulation is known as LNP3, and shows higher transfection efficiency compared to LNPs composed of just DOTAP alone (Simberg et al, 2004). DOTAP is also used in mRNA delivery for protein replacement therapies and antiviral therapies (Simberg et al, 2004).
In CAR_Ma, DOTAP is a feasible and efficient cationic lipid in gene delivery, and is used to provide higher transfection efficiency.
Tween 80
Tween 80, known as polysorbate 80, is a non-ionic surfactant and emulsifier used in various industries, such as pharmaceuticals, food, and cosmetics. It is derived from sorbitol and oleic acid, and is characterized by its ability to reduce surface tension, stabilize emulsions, and enhance the solubility of hydrophobic compounds.
In the formulation of nanoparticles, Tween 80 has become a critical component due to its unique properties best suited for nanoparticle drug delivery. Researchers in synthetic biotechnology utilize Tween 80 due to its surfactant properties that enhance stability and functionality. Tween 80 is frequently incorporated into lipid nanoparticles designed for delivering nucleic acids such as mRNA or pDNA, as it improves the stability of lipid nanoparticles in biological environments, limiting the chance for endosomal escape, while facilitating and prolonging cellular uptake (Sun, 2004). Tween 80 is also able to enhance targeting abilities, especially for delivering drugs across the brain-blood barrier. The coating of Tween 80 aids nanoparticle interactions with the brain’s microvascular endothelial cells, allowing a better cellular uptake and transportation into the brain (Sun, 2004). Studies have also demonstrated that Tween 80 exhibits better drug encapsulation efficiency, and sustained release to those without it (Das, 2012).
In CAR_Ma, Tween 80 is used for aggregation prevention, and promotes electrostatic repulsion to enhance the stability of our formulation.
Nanoparticle Functionalization
In CAR_Ma, nanoparticles both lipid and protein based have ideal properties that allow for the in vivo delivery of the multi-modal RNA vector. However, nanoparticles themselves are quite indiscriminate when it comes to targeted tissue transfections. To counter this, our nanoparticles will be functionalized with anti-CD14 antibodies or nanobodies due to CD14’s characteristic as a monocyte and macrophage biomarker (Ziegler-Heitbrock & Ulevitch, 1993) Nanoparticles offer a vast surface area for the conjugation of antigen recognition domains and are capable of delivering payloads to the targeted cells. The exploration of functionalized lipid nanoparticles with antibodies or antibody-nanoparticle conjugates for cancer drug therapy is not novel (Kumari et al., 2023; Marques et al., 2023). Taking this established principle, we are able to develop antibody-nanoparticle conjugates capable of delivering RNA payloads to transfect macrophages in vivo creating an efficient and safer route for immunotherapy.
References
Ahmad, Javed. “Lipid Nanoparticles Based Cosmetics with Potential Application in Alleviating Skin Disorders.” Cosmetics, vol. 8, no. 3, 7 Sept. 2021, p. 84, https://doi.org/10.3390/cosmetics8030084
Chidambaram, Moorthi, et al. “Nanotherapeutics to Overcome Conventional Cancer Chemotherapy Limitations.” Journal of Pharmacy & Pharmaceutical Sciences, vol. 14, no. 1, 16 Feb. 2011, pp. 67-77, journals.library.ualberta.ca/jpps/index.php/jpps/article/view/9199, https://doi.org/10.18433/J30C7D
Das, Surajit, et al. “Are Nanostructured Lipid Carriers (NLCs) Better than Solid Lipid Nanoparticles (SLNs): Development, Characterizations and Comparative Evaluations of Sun, Wangqiang, et al. “Specific Role of Polysorbate 80 Coating on the Targeting of Nanoparticles to the Brain.” Biomaterials, vol. 25, no. 15, July 2004, pp. 3065-3071, https://doi.org/10.1016/j.biomaterials.2003.09.087
Khan, Shadab, et al. “An Overview of Nanostructured Lipid Carriers and Its Application in Drug Delivery through Different Routes.” Advanced Pharmaceutical Bulletin, 18 Sept. 2022, https://doi.org/10.34172/apb.2023.056
Kumari, M., Acharya, A., & Krishnamurthy, P. T. (2023). Antibody-conjugated nanoparticles for target-specific drug delivery of chemotherapeutics. Beilstein Journal of Nanotechnology, 14, 912-926. https://doi.org/10.3762/bjnano.14.75
Marques, A. C., Costa, P. C., Velho, S., & Amaral, M. H. (2023). Lipid Nanoparticles Functionalized with Antibodies for Anticancer Drug Therapy. Pharmaceutics, 15(1), 216. https://doi.org/10.3390/pharmaceutics15010216
Saha, T., Fojtů, M., Nagar, A. V., Thurakkal, L., Srinivasan, B. B., Mukherjee, M., Sibiyon, A., Aggarwal, H., Samuel, A., Dash, C., Jang, H. L., & Sengupta, S. (2024). Antibody nanoparticle conjugate-based targeted immunotherapy for non-small cell lung cancer. Science Advances, 10(24). https://doi.org/10.1126/sciadv.adi2046
Vitulo, M., Gnodi, E., Meneveri, R., & Barisani, D. (2022). Interactions between Nanoparticles and Intestine. International journal of molecular sciences, 23(8), 4339. https://doi.org/10.3390/ijms23084339
Zheng, Bin, et al. Delivery of Antisense Oligonucleotide LOR-2501 Using Transferrin-Conjugated Polyethylenimine-Based Lipid Nanoparticle. Vol. 39, no. 4, 1 Apr. 2019, pp. 1785-1793, https://doi.org/10.21873/anticanres.13285
Ziegler-Heitbrock, H., & Ulevitch, R. (1993). CD14: Cell surface receptor and differentiation marker. Immunology Today, 14(3), 121-125 https://doi.org/10.1016/0167-5699(93)90212-4