Since it is one of the most fatal diseases, cancer has always been so destructive and problematic for human health. Older cancer treatments are mostly surgery, radiation and chemotherapy. Such therapies have severe side effects, are recurrent, and are drug resistant. Cancer immunotherapy (CIT) has taken center stage in clinical cancer treatment. It mainly uses immunological principles and techniques to stimulate and stimulate the activity of human immune cells, to stop or kill tumor cells. The benefits of CIT over conventional chemotherapy and radiotherapy are: large curative effect, less toxic and side effects, and low recurrence. There are currently several types of tumour immunotherapy that have been trialled in clinical practice: monoclonal antibodies, adoptive cell transfer, immune checkpoint blockade and vaccines. Although immunotherapy is now a viable approach to clinical cancer treatment, it works only in a few patients and can induce autoimmune side effects in patients while the patient is receiving the therapy: immune cells destroy healthy tissue. Immunosuppressive tumour microenvironment (TME) - the main mechanism inhibiting the effectiveness of immunotherapy - is involved principally in activation and infiltration of CTL. Additionally, tumor heterogeneity and tumour drug resistance are very troublesome for tumor immunotherapy. Most immunotherapy drugs - peptides, monoclonal antibodies, genes, cells - are macromolecule drugs. How to get these drugs to the tumour and how to sustain drug activity to make them work is also an issue to be solved in immunotherapy. Therefore, safer and easier tumor immunotherapy that stimulates anti-tumor immune response to maximize treatment efficacy of malignant tumours must be designed immediately. As nanotechnology and nanomedicine have rapidly advanced, nanodrug delivery systems (NDDS) are now increasingly recognised as an approach to increase drug delivery effectiveness and tumor immunotherapy. It can enhance not just hydrophobic drug solubility and drugs' bioavailability, but also prolong drug circulation time in body and enhance drug distribution in body. NDDS can also achieve the in vivo delivery of immunotherapy biomacromolecule drugs, and the co-delivery of several immunomodulatory molecules. While NDDS can be selectively mapped to tumor tissues through different surface modifications (eg, antibodies, peptides, etc.), biological barriers are hard to breach and inevitable immunogenicity are the two main barriers that prohibit NDDS from treating tumors in clinical settings. Bionic delivery systems have appeared, inspired by living organisms in nature, and become popular. In biomimetic delivery technology, endogenous components extracted and purified from human, animal and microorganism tissue are combined with the drug carrier to create new biomimetic drug delivery systems (BDDS). The cells as organic carriers of proteins and molecules in living things are highly nontoxic and nonimmune. The BDDS based on the cell architecture could be an excellent alternative to conventional NDDS and could be applied in the context of tumor immunotherapy. If we paint the cell membrane with NDDS, or if we incubate living cells with NDDS in order to build drug-loaded cells, then the good physico-chemical stability of NDDS is mingled with the biology of natural cells.
Biomimetic Drug Delivery Device Builting
Separation of cells and cell membrane extractions Extraction of cells from whole blood is the most common form of extraction: RBC, white blood cells, platelets and monocytes; immune cells (neutrophils, macrophages and NK cells) are mostly extracted from bone marrow; tumor cells are separated and isolated using cell line culture; stem cells are usually isolated and isolated from animal tissue. Today, peripheral blood mononuclear cells are sorted and separated by the polysucrose (Ficoll) density gradient centrifugation technique. This is done using centrifugation through polysucrose-diatrizoate layering liquid of the concentration 1.0770.001. Red blood cells and granulocytes have high specific gravity and sink to the bottom of the tube upon centrifugation; lymphocytes and monocytes are less specific gravity than or equal to the layering solution, and drift on the surface of the layering solution upon centrifugation. You may see a few cells floating in the layering solution. So too does the Percoll discontinuous density gradient sedimentation method for the purification of lymphocytes. Percoll consists of a particle of silica gel with vinylpyrrolidone - a solution that is capable of reaching a density of up to 1.3g/mL. Pre-mixed density gradient can give good cell separation result within a few minutes by centrifugal force low. Percoll is also widely used to deseed subcellular, bacteria and viruses, damaged cells and slivers from living cells. Ultracentrifugation is the most popular technique to exosomes (EXO). It usually takes multiple centrifugation operations: for example, removing cell debris with a low centrifugal force (300g), and precipitating and preconcentrating exosomes with a high centrifugal force (100000g).
Figure 1. Biomimetic nanoparticles for tumor immunotherapy. (Yu H, et al.; 2022)
There are basically two main phases for cell membrane coating nanoparticles: cell membrane removal and membrane-core fusion. Removal from cell membranes centrifugates components that are not needed in cell culture fluid or plasma, then mechanically or chemically annihilates the cells so that cell membranes and other organelles are isolated, and is then filtered through differential centrifugation or gradient centrifugation. It’s a little different for the membrane extracting of anucleated cells and nucleated cells. In the case of anucleated red blood cells and platelets, the cells are centrifuged from the entire blood, then the red blood cells are lysed with hypotonic solution, and platelets are usually lysed through recurrent freeze-thaw cycles. After lysed cells, they are spun through high-speed centrifugation and then red blood cell or platelet membrane is taken out. Protease inhibitors or phosphatasese inhibitors are generally added to the buffer (pH 7.0-7.4) for membrane elution to keep the membrane proteins biologically active and the cells are kept at 4°C. Nucleated cells (immune cells, cancer cells, stem cells) take a little bit longer to membrane-separate, and more cells are needed for cell membrane separation. The cells are hypotonically lysed or ultrasonically emulsified first to obtain a mixture of cell membranes, intact cells, organelles and intracellular biomacromolecules, then differential centrifugated or discontinuous sucrose gradient centrifuged to yield cell membranes. These removed cell membranes are rinsed in plasma buffer and run through a porous polycarbonate membrane to obtain cell membranes of some size.
Drug Loading Process
Currently, cell-molecular-shielded nanoparticles are just a core-shell configuration. Once you have purified membranes and core nanoparticles, you coat the cell membranes with nanoparticles using a number of techniques. Physical extrusion is one of the most commonly employed techniques. Nanoparticles and filtered membranes are repeatedly pumped out through nanopores of polycarbonate. It is the membrane structure, broken by mechanical stress, that is rebuilt on the nanoparticles. Ultrasonic treatment is an acoustic coating method. Cell membranes and nanoparticles, in the wake of destructive ultrasonic waves, fuse together to make core-shell nanostructures. Compared to physical extrusion, ultrasonic treatment doesn't lose material and is easy to scale up. The semi-stable nanoparticle core and cell membrane as well as the asymmetry of the membrane surface charge can be leveraged to achieve a stable core-shell with the right orientation of the membrane. While all the techniques above can be used to make membrane biomimetic nanoparticles, the physical extrusion is time- and labor-consuming and ultrasonic treatment can destroy the core nanoparticles. They're matched by the development of microfluidic electroporation technology. The electroporation microfluidic chip consists of six elements: two inlets, Y-shaped merging channel, S-shaped mixing channel, electroporation zone and outlet. Both the nanoparticles and cell membranes are introduced into the microfluidic chip from the two holes, and absorbed in the S-channel in full. Once it's through the electroporation zone, the electric pulse between the two electrodes can also drive the nanoparticles into the cell membrane vesicles.
Pharmacokinetic Drug Delivery System Based Biomimetic Drug Delivery
While ongoing studies of how and why natural cell membranes form and function, the development of cell membrane-beading BDDS for cancer therapy and diagnosis has become a popular subject in recent studies. By coating the different cell membranes (red blood cell membrane, platelet membrane, immune cell membrane, stem cell membrane, tumor cell membrane, hybrid cell membrane, extracellular vesicle, etc.) with polymer nanoparticles (polymer nanoparticles, metal nanoparticles, Silica nanoparticles etc.) surface, different cell membrane-loaded BDDS with novel properties and roles have been created.
Red blood cells are the blood cells most commonly investigated as they are a simple type, cheap and accessible. A protein made up of iron that is present in the cytoplasm of red blood cells and can bind oxygen. By way of hemoglobin, red blood cells deliver oxygen to tissues. Red blood cells also carry nutrients and metabolic waste products throughout the blood. BDDS with red blood cells as a carrier can help to optimize the pharmacokinetic and pharmacodynamics of drugs and regulate the immune response to drugs. Furthermore, the reprogrammed red blood cells might lead to immune tolerance and drug delivery.
PLT are anucleated cells, from megakaryocytes, that exist in bone marrow with average size of 2-5 m. They are used for coagulation, hemostasis and to keep blood vessels intact. It has a full cell membrane, and there's a sugar coating on the membrane that can pick up plasma proteins and coagulation factors. They created a tumor-killing nanoparticle by attaching platelet membrane-loaded TLR7 agonist resiquimod (R848). The R848-based drug delivery system turns DC and T cells on, stimulating tumor-specific T cell immunity. The platelet membrane coating can improve the nanoparticles' interactions with various cells in the TME, and thus the activity of R848 at the tumor, and permeability in peripheral lymphoid tissue. In mouse colon cancer models, low-dose nanoparticles almost completely suppressed tumor development in mice, enhanced anti-tumour immunity and produced long-term effects on tumor immunity.
Macrophages are naturally occurring immune cells based on monocytes. TAMs - tumor-associated macrophages - are ubiquitous within the tumour immune microenvironment and are tightly associated with the growth, proliferation and spread of tumours. There are two types of TAMs, phenotype M1 and phenotype M2. M1 type among them has been found to stimulate immune response and suppress growth of tumor; M2 type can promote tumor development, specifically: helping to produce immunosuppressive TME, tumor tissue angiogenesis, and tumour cell intrainfiltration, invasion and metastasis. Because macrophages are involved in the genesis and growth of tumours, they have been targeted for cancer therapy and diagnosis.
T lymphocytes are an important part of the human immune system. Once T cells are turned on by APC, they can recognise exogenous antigens and kill them. T cells also release lymphokines and cytokines to support the immune system.
The tiniest number of white blood cells in peripheral blood are neutrophils: between 40 and 60 per cent of all white blood cells in healthy people. As a central component of natural immunity, neutrophils are among the first cells to attack microbes. So neutrophils could also be used as drug carriers to fight cancer. Premeiotic myeloid precursors of neutrophils might facilitate premetastatic niches. Once the tumor niche is established, it attracts neutrophils and CTCs via a network orchestrated by granulocyte colony-stimulating factor (GCSF), and adhesion molecules like lymphocyte function-associated antigen-1 (LFA- 1) L-selectin and vascular cell adhesion molecule-1 (VCAM-1) which latch on to their receptors and move CTCs to the pre-metastasis niche.
Stem cells are self-regenerating immortal cells that can form at least one species of highly differentiated new cells. In immunotherapy against tumours, SCM-capped nanoparticles are common. Researchers designed a SCM-masked PDA nanoparticle with DOX and PD-L1siRNA for targeted bone metastasis in prostate cancer (PCa). Researchers have reported that SCM coating increases the tumor cell uptake and tumor targeting efficiency of nanoparticles, and under the immunity mask of SCM nanoparticles are able to stick to the tumor and actively stop the growth of cancer cells. It's clear from in vivo studies that the nanoparticles circulate in blood longer than free DOX, and therefore accumulate in tumor tissue. It is a promising method to develop biomimetic multifunctional nanoparticles to treat prostate cancer bone metastasis.
The tumour cells are a kind of cancer cell that you can culture in vitro and grow for as long as you like. Given its immune evasion, anti-apoptotic and homologous targeting potential, tumour cell membranes could also be an important element of membrane biomimetic drug delivery systems in tumor immunotherapy. Biomimetic nanoparticles embedded in tumor cell membranes not only preserve tumor antigens but also augment homologous targeting - a huge boon for tumor immunotherapy. So tumor cell membranes as carriers for drugs has been a hot topic in tumor immunotherapy over the past few years.
There are different cell membranes with different biological purposes. When different cell membranes are mixed together, a hybrid cell membrane can result. Hybrid cell membranes are a much easier and adaptable design for membrane bionic nano drug delivery devices. Hybrid films can be prepared through stirring, ice bath or ultrasonic processing and hybrid film-coated nanoparticles can be prepared through physical extrusion, ultrasonic or microfluidic techniques. Previous hybrid membranes made of platelet membranes and red blood cell membranes coated PLGA nanoparticles. The two dyes were able to copolymerise, as fluorescence microscopy showed that the biomimetic nanoparticles did. This shows that the hybrid film coats the nanoparticles and forms a distinct core-shell structure.
References
- Yu H, et al.; Biomimetic nanoparticles for tumor immunotherapy. Front Bioeng Biotechnol. 2022, 10:989881.
- Zhang Y, et al.; Macrophage membrane biomimetic drug delivery system: for inflammation targeted therapy. J Drug Target. 2023, 31(3):229-242.
- Han X, et al.; Biomimetic Nano-Drug Delivery System: An Emerging Platform for Promoting Tumor Treatment. Int J Nanomedicine. 2024, 19:571-608.
