Monday, August 10, 2026

How Very Low Intensity Magnetic Torque Can Overcome Angular Momentum from Molecular Thermal Agitation in Biological Systems: A Possible Classical Approach to the kT Problem

Mini Review

For several decades we have been discussing possible harmful effects of manmade electromagnetic fields (EMFs) on human health, without being able to accurately highlight the existence of an intensity threshold (or frequency, or both) of EMFs not to be exceeded. However, it is a matter of fact that a huge number of studies on several effects on biological systems of EMFs emitted by human activities at very low intensities and regardless of the frequency of the field have been published so far. Furthermore, the fact that these effects have been significantly detected and quantified even on very simple organic systems such as proteins and cells leave no doubt as to the existence of such effects [1- 5]. Nevertheless, some researchers have pointed out that these effects do not find any theoretical explanation despite having been observed experimentally. In this regard, I would like to begin by citing the paper of Pierre and Dobson [6] in which it is expressly reported that the Lorentz force exerted on ions passing through cells membrane channels due to an applied EMF is negligible with respect to the forces that are needed to the activation of ions gating channel. In that paper the authors considered Na+ ion moving in cell channel at velocity v under a magnetic field (MF) B. Following Lorentz’s law, this charge should be subjected to the force F = qv× B .

The authors highlighted that a MF of 3 x 108 T is needed to induce a force of 1 pN and even considering ion velocity of 102 m s-1, a MF of 105 T would be necessary, which is several orders of magnitude larger than any magnetic field on Earth! Another study on the apparent contradiction between experimental observations of the effects of EMFs and negative theoretical results is that of Adair [7]. He made a simulation in which Hemoglobin (HB), chosen as protein prototype, is exposed to an EMF with amplitude E = 100 kV/m for Δt = 1 ns (the time integration from Sandia electric pulse, corresponding to the frequency of 900 MHz). The EMF will generate a torque on the protein because its significant dipole moment [8], inducing an angular impulse which has to compete with the mean angular momentum from thermal agitation to which the protein is subjected too. In this simulation, despite of the large value of amplitude of the EMF which was used, the mean angular momentum from thermal agitation resulted significantly larger than the impulse from EMF. Why do theoretical simulations contradict experimental observations? On the other hand, how is it possible that all the experimental results observed in recent years concerning the effects of EMFs are wrong? This apparent contradiction has been named the “kT problem”.

Let we consider macroparticles with a large dipole moment such as charged dust particles, mitochondria and some cells under exposure to an EMF. They will align towards the direction of the applied field: this phenomenon was named in literature “pearl chains” [9-13]. It can be easily explained by classical theory because the large mass of the organic particle gives rise to a large angular impulse that will overhead mean angular momentum from thermal agitation, as it can be easily shown. In contrast, kT problem arises when dealing with particles at the microscopic level such as molecules and ions. In this case, classical physics cannot longer be used, but a quantum approach should be used [14-23]. Indeed, light particles such electrons should have large wavelengths so that they can move from one side of a potential-energy barrier to the other side, despite of not having enough kinetic energy to overhead the barrier. However, in the second simulation above reported a macromolecule was taken in account so that even using classical physics a correct explanation should be found.

That is, it is a situation which is a cross between an atomic particle and a macroparticle, so that a semiclassical approach could apply. Instead, in the simulation reported above it seems that this cannot be done only because an adequate exposure time to EMF was not taken in account. For instance, in the first simulation reported above the integration time was not really considered and a constant intensity of MF was assumed. Nevertheless, even if the MF intensity has been assumed to be constant, the effects due to the applied MF should also depend on exposure time. Even if the torque induced by the applied MF is not intense enough to produce a displacement of the organic particle, however it will produce a very small displacement that will change the initial conditions from which the subsequent action of the force will begin in the following instants of time, In such a way that these imperceptible shifts will add up as the particle is exposed. An illustrative example of this situation can be represented by a (constant) force produced by the impact of a drop of water on a rock that does not produce any visible effect. However, the persistence of this action over time will produce considerable macroscopic effects on the rock that seem inexplicable by the single event, but which are instead produced by the sum of many events of this type. Regarding the second simulation above reported, instead, a time interval given by Sandia electric pulse Δt = 1 ns was taken in account, corresponding to the EMF frequency of 900 MHz. This choice depends on having considered a typical high frequency (HF) EMF. In this scenario the protein exposed to the EMF rotates from one side to the other following the frequency of the field, returning to the initial position after 1 ns, so that the average displacement is zero and no effect induced by the EMF could be explained.

However, this reasoning is correct if the particle rotates in a vacuum, whilst if it is embedded in a medium it cannot follow the EMF HF oscillation due to the inertia of particle and to the friction induced by the viscous damper of the medium in which the molecule is embedded. Hence, the particle exposed to the EMF will undergo a non-zero displacement correlated to the exposure time and a time interval larger than that of Sandia electric pulse should be taken in account. In the study published in [24] a macrodipole represented by the α-helix of typical proteins was considered to test this model, explaining the rotation of proteins α-helices and their alignment along the direction of the applied EMF observed by FTIR spectroscopy. This scenario can explain all the effects of exposure to EMFs of biological systems observed so far. Indeed, α- helix is present in all biological membrane channels and constitutes the walls of these channels [25-27], so that an applied EMF at very low intensity could not induce the rotation of these α-helices considering an integration time of the order of Sandia pulse, due to the significant binding forces in membrane channels.

In contrast, the impulse of the magnetic force due to an applied EMF calculated for the duration of exposure time can induce a torque on the α-helix causing a little increase in the diameter of cellular membrane channel [28-30]. This increase doesn’t have to be huge. A small variation is enough to produce an alteration in the flow of ions through cell membrane channel, causing alterations in cellular functions and the consequent effects that researchers have so far observed. Otherwise, it is true that the α-helix displacement must also compete with thermal molecular agitation, as Adair showed in his study [7]. However, even in this sense, considering an integration time comparable to a typical exposure time, it is easy to demonstrate that the magnetic torque exceeds the angular momentum from molecular thermal agitation [29,30]. A further study to explain experimental results of exposure to EMF by classical theory was carried out by the author and reported in [31], in which the competition between the mechanical moment due to a MF applied to HB protein and the diffusion quantity which is due to thermal molecular agitation was quantified. The complete equation of motion of a molecule subjected by both actions is given by

in which f(φ) is a distribution function and Φ is the rotational diffusion coefficient. The first two terms of this equation are due to the equation of diffusion process, the last term represents the quantity due to the mechanical moment and ω is the angular velocity of the molecule that can be calculated by the term

in which mH is the mechanical moment, FH is the force induced by the MF, r is the radius of the molecule and d = r sinφ is the distance between the application point of FH and the center of the molecule [32]. At the equilibrium, the torque of MF should balance thermal agitation so that we should have

The solution of this equation is given by

and the number of molecules that are rotated by an angle φ with respect to the direction of the field is given by

in which N is the total amount of paramagnetic molecules per unit of mass, μ is the molecular magnetic permeability and H is the applied MF [32].

The ratio of the numbers of molecules rotated with respect to the direction of the MF by two different angles φ1 and φ2 at the corresponding temperatures T1 and T2 is given by:

The parameters in the study of [31] were used to test the modeling above reported. As a result, the terms 2kBT1 and 2kBT2 can be neglected in comparison to μH and it can be assumed that cos φ1 = cos φ2 given that the displacement of molecules at the temperature T1 should be close to that at the temperature T2.

Finally, Eq. (6) can be written

so that the ratio between the number of molecules that are oriented along the direction of the applied MF by an angle φ is inversely proportional to the ratio between the temperatures. Such as showed in previous literature concerning FTIR spectroscopy, the intensity of the vibration band Amide I should be proportional to the number of proteins α-helices aligned with the direction of the applied MF. The ratios between the Amide I integrated area ratios of exposed/unexposed HB samples and some couple of temperatures T1 and T2 were computed in [31], providing the result that these values coincide with those obtained from Eq. (7), proving that the alignment of α-helix with the direction of an applied EMF is inversely proportional to the temperature. Above all, it was proved that the rotation of α-helix is not impeded by the Brownian motion due to thermal molecular agitation despite of the low intensity of MF used in [31], in agreement with the theoretical classical approach above reported.

Conclusion

Previous studies have highlighted that the effects caused by exposure of some organic systems to man-made electromagnetic fields at low intensities cannot be explained theoretically because angular momentum from molecular thermal agitation of surrounding medium is found to exceed the torque induced by EMF. Some authors assumed that a different approach by quantum physics is needed to solve this problem, in particular if we consider particles at atomic or subatomic size scales such as electrons or ions crossing cell membrane. However, if larger scales are considered, such as macromolecules like proteins, then classical physics can also be able to explain experimental results observed after exposure to man-made electromagnetic fields.

BJSTRAngelaRoy

Thursday, August 6, 2026

Antimalarial Aloe Compounds

Introduction

Among the most prevalent diseases caused by protozoan parasites, malaria is caused by parasites of the genus Plasmodium and transmitted to humans by infected female anopheles’ mosquito [1]. Plasmodium falciparum, Plasmodium malaria, Plasmodium knowlesi Plasmodium ovale, and Plasmodium vivax are the five parasite species that cause malaria in humans. Among these species, P. falciparum causes the most severe form of malaria and hence higher mortality rates [2]. The estimated number of malaria deaths stood at 627, 000 of the estimated 241 million cases of malaria in 2020 worldwide [3]. To control malaria, many popular practices exist to avoid the nuisance of mosquito bites such as fumigation, burning green leaves on the hut’s threshold, mosquito coils, insecticide sprays, and repellents. Smoke is a common method of repelling biting mosquitoes that is used throughout the world from ancient times to current [4]. To treat infectious diseases such as malaria, herbal medicine plays a great role. Due to the vast metabolic diversity of plants, natural products may be an alternative treatment opportunity of cheap and easy to treat malaria infection. For instance, artemisinin and quinine are antimalarial drugs that were isolated from plants. As a result, it can be generalized that plants have potential as sources of active chemical components used for antimalarial drugs [5].

Plants are considered to be the richest resource of active molecules in traditional systems of medicine and modern medicines. The use of plants and plant products both in medicines and as medicine could be traced as far back as the beginning of human civilization [6]. In human history medicinal plants, a source of remedies, are widely used as alternative therapeutic tools for the prevention or treatment of many diseases [7]. The genus Aloe is one of the top medicinal plants that have obtained its popularity from time to time. Aloe plants are used from immemorial time to nowadays. Therefore, they become a popular household remedy exhibiting a range of beneficial health-promoting properties. Aloe in one form or another is a common domestic medicine and is the basis of most pharmaceutical preparations [8]. For instance, investigations have led to increased importance of the mostly known Aloe species, A. vera due to its dependable medicinal properties, and it has been used in the preparation of pharmaceutical products [9].

Chemical Compositions of Aloe

Figure 1: Structures of some antimalarial compounds.

Combinations of active molecules extracted from Aloe species have been indicated to confer a variety of biological effects with different mechanisms of action [10]. The chemical compositions that have been identified in Aloe plants include simple and complex polysaccharides, minerals, vitamins, enzymes, hydrocarbons, fatty acids, indoles, pyrimidines, aldehydes, and ketones, dicarboxylic acids, phenolic compounds, phytosterols, and alkaloids with potential biological and toxicological activities [11]. Various components present in Aloe species have been found effective against many diseases, including malaria. In every application of Aloe species, preparing extracts of Aloe parts is mandatory. The leaves of Aloe plants are the most commonly used are heterogeneous and can be divided into three major parts. These are,

1. The majorly consists of structural components of the leaf part, the outer green epidermis.

2. The part that vascular bundles are placed where the bitter latex or sap is obtained, the outer pulp region below the epidermis; and

3. The inner leaf pulp, which consists of Aloe gel and containing parenchyma cells [12,13]. Although leaves are the most used part of the plant, recently some studies have reported the bioactive roots [14] and flowers [15] of the plant. Aloe species have become of great interest to researchers who have tried to identify the compounds responsible for these beneficial effects. Several constituents from various classes such as alkaloids, anthrones, chromones, flavonoids, glycoproteins, naphthalene’s, and pyrones have been isolated from the genus Aloe [16]. Aloin, aloesin, aloenin, aloeresin, aloe-emodin, apigenin, acemannan, and chrysophanol are some examples of such bioactive compounds Table 1 and Figure 1 [17-25].

Table 1: The bioactive compounds in the genus Aloe.

The Antimalarial Aloe Compounds

Anthraquinones

Above all the genus Aloe is rich in anthraquinones which play a crucial role in medicinal applications. Many investigators showed the antimalarial activities of anthraquinones of Aloe species. In the literature, the leaves and roots of different Aloe species were tested and showed variable antimalarial activities. One of the most known anthraquinones, aloe-emodin emodin which was isolated from leaf latex of Aloe, A. macrocarpa Todaro showed potential antimalarial activity against malaria when compared with chloroquine [26]. In addition to this the chrysophanol, aloesaponarin I, and aloesaponarin II are anthraquinones that were isolated from the root of A. pulcherrima showed ant plasmodial activity against both chloroquine-resistant and -sensitive malaria parasites, P. falciparum [27].

Anthrones

The most studied Aloe constituents against malaria were anthrones. Anthrones are large classes of the genus Aloe and they are biologically active. Table 2 shows the specific anthrones of Aloe species against malaria along with parts of the plant [28-30].

Table 2: Antimalarial anthrones of Aloe species.

Chromones

Chromones also showed potential antimalarial compounds isolated from the Aloe species. In the literature, the compound, (E)-2-(1-hydroxy-2-methylpropyl)-8-(6’-O-cinnamoyl)-β-Dglucopyranosyl- 7-methoxy-5-methylchromone (HCGMM) has been reported as is a potential antimalarial compound from A. debrana Chrstian. When compared to the mice in the negative control group HCGMM showed significant suppression (p<0.05) against P. berghei at dose levels of 25, 50, and 100 mg/kg/day [31].

Other Compounds

The potential of Aloe species means the molecules in them have wide applications in combating malaria. From A.otallensis, naphthalene derivative identified as 2,8-O,O-di(β-D-glucopyranosyl)- 1,2,8-trihydroxy-3-methylnaphtalene (plicataloside) evaluated for its in vivo antimalarial activity using a 4- day Plasmodium berghei suppressive test method. The plicataloside (100 mg/kg) inhibited parasite growth by 40.7%. It was proposed that plicataloside may minimize oxidative stress thereby contributing to the antimalarial activity of the plant [32]. The literature shows that some flavonoid derivatives, xanthones, stilbenes, coumarins, lignans, tannins, quinones, terpenoids, steroids, and alkaloids possess antimalarial activity [33]. Among these, natural, semi-synthetic, and synthetic quinones are effective antimalarials [34]. Several investigations showed that many naturally occurring compounds possess antimalarial activity when tested in different malarial diseases [31].

Conclusion and Future Aspects

Based on the indigenous antimalarial effects of Aloe plants, the scientific studies, in vitro and in vivo reports confirmed that the leaves and roots of Aloe plants possessed genuine antimalarial activities which could be attributed to the presence of the active chemical constituents. More, the activity of the active constituents along with their relative margin of safety merit the use of these compounds as leads to the development of safer, cost-effective, and more potent alternative drugs for the treatment of malaria. Considering that natural molecules have acted as natural templates in the development of antimalarial agents, it is encouraged to investigate further analyses into Aloe constituents and their values against malaria. It should be followed with phytochemical and pharmacological analyses in order to give scientific ground to medicinal knowledge and future potential utilization. Therefore, further studies are needed to determine which compound act as a strong antimalarial agent or synergistic effects of the compounds are used for antimalarial activities. In addition, it is recommended to test other parts of Aloe plants like flowers, leaf gel, leaf skin, etc. against malaria. Further analysis is important to identify the result; if malaria drugs like chloroquine are incorporated into Aloe extracts/ isolated compounds. Although most studies are directed towards the curative effect of the test substances, in-depth pharmacokinetic and pharmacodynamic studies are needed to elucidate their mechanism of action.

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Chimeric Antigen Receptor- T Cell Therapy: In Vitro Activation and Comprehensive Expansion of Specialized T-Cells to Neutralize Deadly Cancerous Cells

Introduction

Chimeric antigen receptor based therapy is the most promising and new treatment for cancer. This therapy is very effective in many types of cancer. And the most innovative part of this treatment is that it can be considered the most effective treatment when other treatments get failed. This treatment gives the most satisfying results and the success rate is very high. This process is directly linked to the immune system of the body. It is a type of cell based therapy that uses the T cells of the immune system, collectively worked in the lab to get hold of cancer cells and destroy them. This treatment involves the alteration of genes inside the T cells resulting in expression of unique cluster of differentiation which identify and investigate tumorigenic cell surface receptors providing immunophenotyping of cells. This therapy is highly used to attack the dangerous cancer cells. Our immune system has the most complicated system yet the simplest one. Our immune system has special powers to identify the foreign substances that usually enter inside the system. This process starts with, where the body finds the antigens that is present on the surface of cells. T cells are very specialized cells of the immune system, they have their specialized proteins. These proteins get attached to the foreign substances and this helps in triggering rest all parts of the immune system, which helps in killing those foreign substances. To better comprehend CAR T-cell therapy, first we have to perform more extensive research about T cells. Lymphocytes, which are white blood cells, play a crucial role in battling infection and disease, including cancer. There are various types of lymphocytes. T cells are one sort of cell.

T cells have special powers, so they travel all through the body looking for and getting hold of damaged cells. Whenever we come in contact to a new illness or disease, our body automatically produces certain amount of T cells to fight with that infection or disease. The mechanism of our body is very clever, so it stores some in reserve mode so that if we again come into contact with the virus, our body will definitely recognize it and kill it right away with very less time from the earlier ones. T cells are very effective at fighting infection. However, during cancer it becomes very difficult for them can to discriminate between a cancer cell and a normal cell. As a result, cancer cells sometimes go unnoticed and unobserved. T cells are being trained in vitro to recognize these deadliest cancer cells by scientists. And one of the novel approaches to this is the CAR cells. These CAR T cells are genetically engineered to get hold of the cancer cells ad help in the treatment. There are certain patient derived immune cells that are designed to express recombinant or chimeric antigen receptors on its surface to recognize and target specific tumor-associated antigens and induce a cell-mediated attack. This ability to reuse T cells has opened up many new techniques for having a very personalized cancer treatment, notably for hematologic malignancies. Furthermore, there is a history of performance of CAR T cells in cancers such as leukaemia and lymphoma, now-a-days there are lots of undergoing clinical trials involved for the treatment of solid tumours. These tumors are quite dangerous and expand rapidly inside the cell. Cancer, from the past years has been established as the main cause of death on a global scale. Several novel treatments and cytotoxic immunotherapies have been developed and commercialized. But till now there has been no complete cure for cancer. It is indeed an epidemic disease in nature.

Cancers has a very complicated behaviour and it involves several genetic and cellular variables in tumorigenesis and metastasis, successful immunotherapy that targets tumors at both the cellular and genetic levels are required. And to revolutionize the whole process, CAR T cell therapy has proven to be a novel approach. In this, the T cells which are obtained from patient blood are altered in vitro to express artificial receptors targeted to a specific tumor antigen. These are able to directly detect the tumor antigen without using the major histocompatibility complex. The application of this therapy in almost all types of cancers has proven to be very effective. This is because; T lymphocytes are genetically engineered to stand as a strong fighter in front of deadliest cancer cells. This novel therapy is known as immunotherapy, gene therapy, or cancer therapy. The human defence system is capable of distinguishing between self and non-self-molecules, such as bacteria, viruses, and aberrant cancer cells. There are certain principles on which human immune system works. Based on the principle of antigenicity sand immunogenecity, tumor cells can be identified with little bit detailing of foreign substances. Cancer cells, on the other hand, are quite clever enough in manipulating our whole immune system for their advantage. These cells make the symptoms quite hard to get noticed in the early days. As a result, situation gets out of control at the later stages. Immunotherapy commonly called as biotherapy has some scientific insights that make it naturally capable of recognizing infections and malignant cells. Immunotherapy has emerged as a major treatment option in recent years. Depending on the condition, each treatment method has advantages and downsides. With an increased understanding of the immune system, several novel immunotherapies are being explored, including techniques for stimulating the immune system to work impertinently to target cancerous cells. CAR T cell therapy has been most successful in treating hematological malignancies; however, research is still going on its efficacy against solid tumors.

Car T Cell Therapy

CAR-T cell therapy has shown promising results for a number of malignancies. This is a medical procedure that engineers T lymphocytes to kill tumor as well as cancerous cells. The initial step in this therapy is to isolate a patient’s peripheral blood. Through the process of apheresis, blood from patients is extracted, divided into its component parts, and genetically modified with CAR construct before being reinserted back into the patient’s body. Apheresis is now a common technique used by blood banks to separate platelets and other blood components for the treatment of a number of illnesses, including hematologic and renal issues. It is acknowledged as a risk-free approach for both healthy people and sick as a result.

Challenges Involved in the Process of Car T Cell Therapy

Although CAR treatment has emerged as a potential anticancer method, it is still not waived off from the problems. The improved performance and efficacy profile office hours CAR T cell treatment are important areas of research that require long-term clinical trial follow-up. Furthermore, CAR T cell therapy has been linked to a number of serious side effects, including neurological toxicity, cytokine release syndrome (CRS), B cell aplasia, tumour lysis syndrome, and allergy. When CAR T cells are multiplied in the body, it helps the other cells to create cytokines that helps in destroying the cancer cells. Few known side effects are fatigue, nausea, headache, fever, and chills, as well as a drop in blood pressure, tachycardia, and capillary leakage. Another uncommon result is CAR T cells affects the metabolism of B cells resulting in aplasia.

Mechanism Involved in the Process of Car T Cell Therapy

The ability of chimeric receptors to fuse or split discrete critical activities, such as identification, co-stimulation, and activation, in separate chains of a receptor molecule expressed on a cell surface by replicating the complexity of the original T cell receptor structure, distinguishes them. T cells are proved to be very advanced and complicated cells of our body which generates maximum iummunity to fight against fatal diseases. It basically doesn’t require any outside formulation for the activation and proliferation of cells. This technique is indeed the most natural process in an artificial treatment. As this treatment uses one’s own immune system to proliferate and perform its mechanism. T cell activation and proliferation require the presence of stimulatory molecules, which also aid in CAR T cell cytokine production. This technique uses an engineered chimeric receptor that works with scFv fragments for T cells. In comparison to other small functional compounds, this mechanism improves the capability of T cells. This mixture of a ‘living medication’ and artificial technique in the immune system fights cancer. Furthermore, CAR T cells can survive in the body as long-term memory cells for several years. Whenever there is any chance if relapse of cancer in the cells, these CAR T cells that has been stored as memory cells helps in the detection and destroying the cancer cells. Another benefit of CAR T cells is that they specifically target tumour cells rather than auto antigens. As a result, it is non-lethal to host cells. Once produced on the surface of an engineered T cell, the synthetic immunoreceptor’s scFv selectively binds to target antigens expressed on a cancer cell.

CAR consists of extracellular antigen-binding domains derived from monoclonal antibodies and intracellular signalling domains connected together by a hinge and a transmembrane domain. The CD3 chain of the T cell receptor complex is present in first-generation CARs, but second-generation receptors have one stimulatory molecule (CD28) and third-generation CARs contain two stimulatory molecules, such as CD28 and OX40. CARs are typically delivered into primary T cells using a vector. CAR-T cells’ cytotoxic effector activity is often maintained by CD3. CD28 (blue) has a role in T cell proliferation and cytokine production. The intricacy of each CAR generation grows due to the presence of costimulatory molecules or the triggered promoter for CAR, chimeric antigen receptor; scFv, single-chain variable fragment; IL, interleukin and transmembrane. The mechanism of genetically engineered CAR T cell is very simple yet the most complicated one. It indeed takes huge research to design a specific cell. And this deadly combination of in vitro and in vivo takes lots of time to be perfect and get inserted inside the host cell. The most interesting part of it is, the efficacy depends on the immune system.

What are the Various Modes of Delivery in this Treatment

Gene therapy works on various methods for the introduction of DNA into cells. The most common method employs recombinant viruses (also known as viral vectors), biological nanoparticles, and non-viral methods based on naked DNA delivery.

The Viral Vector Gene Delivery

In gene therapy, viral vectors such as -retrovirus, lentivirus, and adenovirus vectors are commonly employed. One of the most frequent ways of gene therapy is the retrovirus transduction. In this method a reverse transcriptase is used to enhance the integration of artificial genes inside the host genome. These Retroviral vectors have an inbuilt potential to disrupt the genomic region, resulting in neoplastic transformation. As a result, retroviral vectors play a vital role in the process of gene therapy. There are certain lentivirus that have proven the best in expressing cells of both nature that is dividing and non-dividing cells, which as a result helps a lot in the in vivo and in vitro methods.

Few Methods of Non-Viral Delivery

4.1.1. Transposon Conjugation: Transposon method is a very unique method and it differs greatly from rest others. Transposon delivery is a non-viral technique that combines transposon DNA and a transposase enzyme to convey stable genes. In this process the two important vectors are PiggyBac (PB) and sleeping beauty (SB). Transposons use a cut-and-paste technique to move from one gene location to another.

Electroporation

Electroporation has evolved as an effective method for changing the genes of several cell types. In this process, Electric fields are used to temporarily damage the cell membranes of the target cells. As a result, it helps the charged molecules to enter inside the cells. Human T cell electroporation has been linked to a 40-60% increase in gene expression and an increase in cell viability of up to 80%. The low transfection efficiency and redundant cell injury are two of the disadvantages of electroporation.

Advancements in Car T Cell Therapy

There are Many Advanced and New Versions of CAR T Cell

a. Tandem Car: A single intracellular domain is coupled to a single CAR structure that targets two tumour antigens and, more significantly, its own antigen recognition domain. Researchers are aiming to create a complex technology known as a Tan-CAR, which consists of two distinct antigen recognition sites that are connected by a linker, placed next to one another on an intracellular domain, and produced as a single CAR on the cell surface.

b. Bispecific Car: That is targeting T cells, two CARs are generated simultaneously, each of which has two distinct antigen-recognition domains that target two tumour antigens. When this CAR interacts with two tumour antigens, it can start a cascade of effector chemicals that works synergistically.

c. Crispr Cars: CRISPR is a gene-editing method that modifies a DNA sequence using a guide gRNA. This approach offers a dependable and skilled gene knock-in process as a gene integration technique. The use of CRISPR technology in immunotherapy might be beneficial. CRISPR has been extremely important in the scientific community nowadays.

Advantages of Car T Cell Therapy

The quick turnaround time, single CAR T cell injection, and successful outcomes of CAR T cell therapy over other cancer treatments are its biggest advantages. Additionally, the patient only needs cautious monitoring and care for two to three weeks. Because CAR T cells may stay in the host body for extended periods of time and have a consistent capacity to identify and eliminate cancer cells during recurrence, they are regarded as the muchneeded treatment for cancer, and their efficacy may endure for decades. The use of CAR T cell therapy in those who are unable to receive a transplant has been authorised in the modern world. The CAR T cell therapy has been developed to offer an alternative to other transplants [1,2].

CAR T Cell Generation Flowchart

Flowchart 1.

Flowchart 1.

Protocol for CAR T Cell Generation

a. Isolation of Peripheral Blood mononuclear cells:

• Directly separate lymphocytes from whole Blood.

• Density gradient centrifugation of whole blood sample helps to identify different layers, which signifies the concentrated RBC deposited at the bottom part whereas various WBC and platelets lies in middle.

• Discard the plasma/ water part from the tube after a complete centrifugtaion and collect the platelet part.

• Add a mixture of biotinylated monoclonal antibodies against the non-CD8+ and non-CD4+ T cells to the sample followed by incubation.

• Add MyoneTM SA Dynabeads, which will bind to the antibody labelled cells during short incubation.

• Separate the beads bounded cells on a magnet and discard.

• Remaining sample is now ready to carry forwarded for further process.

b. Activation of T cells:

• Use T Cell TransAct™.

• Lentiviral transduction of activated T cells with a CAR gene construct.

c. Expansion of activated T cells:

• Use serum media to initiate T cell propagation.

• Scale up and exppansion with TexMACS medium (Adapted from serum suspension).

d. Phenotyping and functionality test:

• Use Immunophenotypic markers in T cells.

• Flow cytometry for immunophenotyping.

Conclusion

CAR T CELL therapy has indeed proven to be the most successful treatment in treating cancer. The success rate is quoting high in this process. This technique has proven to be a boon in amidst of dangerous disease. Researchers have got many benefits through this therapy. And still, many researches are going on to get further advancements in this therapy. Cancer, being the most deadly disease, Car T Cell has been a life savior which uses immune cells to get solutions of cancers a highly effective technique that needs more novel innovations. CAR T cells are offered in a variety of scientific frameworks, which can vary greatly between countries. The combination of obstacles and technology necessitates uniformity; yet, CAR T cells provide patients with hope for advanced treatment. Because the first therapy is already on the market, there is a chance that a more precise and improved alternative will become accessible in the coming decades.

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