The Drug Delivery System—liposome (part two)

1.1.2 Drug Ion Electrical Properties and Drug Loading Capacity

The interaction between the drug and the phospholipid layer molecule has an important effect on the structure and load of the liposome, and the effect of the charge effect is particularly significant. Generally, when the charge properties of the drug and the phospholipid molecular layer are the same, it is not easy to be encapsulated. By adding appropriate excipients during the preparation of the liposome to make it a charged liposome opposite to the charge of the encapsulated drug, the drug encapsulation rate can be improved. For example, in the preparation process, octadecylamine is added to obtain positively charged liposomes, and phosphatidic acid is added to obtain negatively charged liposomes. The antiviral drug cidofovir is negatively charged under normal physiological conditions. It is found that liposomes made with positively charged phospholipids composed of DOTAP and DC have a significantly higher encapsulation rate than liposomes made with electrically neutral phospholipids. However, other studies have shown that when the positively charged drug sumatriptan uses a neutral phospholipid as the membrane material, the encapsulation rate is low, and when the positively charged material stearylamide is added to the membrane material, the phospholipid membrane is significantly strengthened, thereby t                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          lop09he leakage of the drug is prevented, and the drug load is increased. However, the encapsulation efficiency was lower than that of positively charged membranes after the addition of negatively charged membrane dicetyl phosphate, indicating that in some drug encapsulation processes, charge is not the main factor influencing.

1.1.3 Decoration and Encapsulation Rate of Medicinal Chemical Structure

The chemical structure of a drug determines its physical and chemical properties. By modifying the structure of a drug to some extent, the hydrophilic and hydrophobic properties of the drug can be improved, thereby improving the encapsulation efficiency of the drug. Researchers reacted the anticancer drug cyclocytidine with palmitic acid to obtain two derivatives of monopalmitate and dipalmitate, which were separately encapsulated to prepare liposomes. As a result, the encapsulation rates of the two derivatives were found. It rose to 86.5% and 93.7% respectively, while the original drug was only 21%. The HLB value shows that on the one hand, the original drug is converted from hydrophilic to lipophilic after esterification, and the encapsulation position of the drug is correspondingly transferred from the aqueous phase to the external lipid phase. On the other hand, the long ester chain obtained by structural modification can be embedded in the lipid membrane. In addition, the fluidity of the lipid membrane is reduced, thereby increasing the liposome stability and encapsulation efficiency.

  1. Liposomal particle size design

Particle size is an important evaluation index of liposomes, and its size and degree of uniform dispersion directly affect the in vivo behavior of liposomes. Large particle size liposomes are easily endocytosed by macrophages and concentrated in the liver. Smaller particle size liposomes can effectively prolong the circulation time of the drug and play a long-lasting effect. When the particle size is less than 50nm, liposomes can penetrate the liver endothelium and enter the spleen, bone marrow and tumor tissues. Duan Yisong et al etc. used long-circulating material polyethylene glycol to prepare mitoxantrone long-circulating liposomes with an average particle size of 60nm. Compared with ordinary liposomes, the average residence time in rabbits was prolonged by 6.2h, reflecting Its long cycle advantage. Awasthi  et al. Investigated the particle size on the circulation time of PEG-modified liposomes in rabbits and found that the optimal particle size is 160-220 nm. Large particle size liposomes (400-530nm) are highly targeted to liver and spleen-enriched reticular macrophages. When the liposome particle size increases to 1-12 μm, it is easily taken up by the lungs. After azithromycin was prepared into cationic liposomes, the mice were administered tail vein to study the distribution of azithromycin in mouse whole blood and various tissues. AUC increased by 7.4 times. This is because liposomes larger than 6 μm will be mechanically filtered by pulmonary capillaries and then taken up by monocytes into lung tissue.

  1. Preparation method selection

The preparation method of liposomes greatly affects the structure and particle size of liposomes, so it is generally selected according to the nature of the drug and the purpose of the drug. For fat-soluble drugs, mechanical dispersion methods such as film dispersion method and freeze-drying method can be selected to prepare multilayer liposomes with large particle diameters, so that the drug is slowly released in the target tissue. If you need to increase the drug’s transport speed, you can choose to prepare small single-compartment liposomes. The main methods include ethanol injection, surfactant treatment, film-ultrasonic method, and so on. The drug loading of water-soluble components is generally not high. The key is to increase the volume of the aqueous phase in the liposome. Therefore, large monolayer or polycystic liposomes are generally selected for preparation. The reverse thin film dispersion method and the double emulsion method and the freeze-thaw method  are suitable for the preparation of large particle size aqueous drug-loaded liposomes. Among them, the reverse thin film dispersion method is mostly large monolayer liposomes, including The sealing rate can reach 60-65%, and some studies have shown that for the hydrophilic drug salvianolic acid B, the large monolayer liposomes (LUV) prepared by the reverse evaporation method, the ethanol injection method, and the double emulsion method are used. Compared with liposomes, the encapsulation rate is the highest. The repeated freeze-thaw process in the freeze-thaw method will be accompanied by the formation of ice crystals, which will cause mechanical damage to the phospholipid bilayer, thereby increasing the chance of water-soluble drugs entering the phospholipid bilayer and increasing the encapsulation rate. At the same time, multi-compartment liposomes can be prepared into small single-compartment liposomes by repeated extrusion and freeze-thaw methods, which increases the drug loading space and drug loading. In addition, the microencapsulation method is suitable for preparation Small-particle-size drug-loaded liposomes in aqueous phase. For amphiphilic drugs such as weak base and weak acid, they can be encapsulated by active drug loading methods, such as PH gradient method, ammonium sulfate gradient method metal ion gradient method, and so on. In some cases, a single method alone cannot meet the requirements of encapsulation efficiency and particle size, especially for compound drugs with different properties in the co-loading concentration, so it is often combined with several methods.

 

Facts about mesenchymal stem cells

Bone marrow primitive mesenchymal stem cells are bone marrow stromal stem cells. They are a subpopulation of cells found in mammalian bone marrow stromal cells that have multiple differentiation potentials to form bone, cartilage, fat, nerves and myoblasts. They not only have mechanical support for hematopoietic stem cells (HSC) in the bone marrow, but also secrete a variety of growth factors (such as IL-6, IL-11, LIF, M-CSF, and SCF) to support haemopoiesis. Mesenchymal stem cells (MSCs) are a type of early undifferentiated cells that have the characteristics of self-renewal, self-replication, unlimited proliferation, and multidirectional differentiation. They can secrete cytokines to reduce inflammation, reduce tissue cell apoptosis, and promote proliferate and perform immune regulation of endogenous stem progenitor cells, so as to achieve the effect of repairing tissues and organs. After continuous subculture and cryopreservation, it still has the potential for multi-directional differentiation, which is called “universal cells” in the medical community.

Functions of mesenchymal stem cells

  1. Multi-directional differentiation: they have strong proliferation ability and multi-directional differentiation potential;
  2. Immunomodulation: they are low immunogenicity, with immunoregulatory function. They won’t cause immune rejection, and can inhibit rejection;
  3. Rich in quantity: they are rich in various tissues and easy to collect.
  4. Strong fertility: up to 1 billion cells can be cultured in vitro for multiple uses.
  5. Safe and reliable: their genes are stable, not easy to mutate, and still have the characteristics of stem cells after multiple passages.
  6. Awide range of application: they have a wide range of applications and can be used to treat almost all tissue damage, aging and degenerative diseases.

Applications of mesenchymal stem cells in humans

  1. The cells are differentiated into functional cells, replacing functional cells that are in decline or injury;
  2. Start the sequential expression of regeneration-related genes, so that endogenous decline of tissues and organs and damage repair are initiated;
  3. Exogenous cells enter the body, secrete a variety of growth nutrition factors, and improve the internal microenvironment of tissues and organs;
  4. Immunomodulation can reduce the inflammatory response of local tissues and organs;
  5. Activate and improve the quality of immune cells and eliminate and prevent hidden diseases
  6. Differentiate skeletal muscle cells, make fat distribution even, muscles firm, muscles and bones soft;
  7. Promote collagen production, a variety of growth factors secreted by cells, effectively regulate the endocrine system and hormone system.

Diseases available for mesenchymal stem cells

① Hematological diseases: mainly for leukemia;

②Nervous system diseases: such as Parkinson’s syndrome, Alzheimer’s disease, traumatic brain injury, spinal cord injury, motor neuron disease, etc .;

③ Parenchymal organ damage or disease: such as cirrhosis and its complications, lung and other tissue fibrosis;

④ Immune system diseases: mainly use the immune regulation of stem cells, such as systemic lupus erythematosus, rheumatoid arthritis, etc .;

⑤ Cardiovascular diseases: such as myocardial infarction, vascular disease, ischemic heart disease, etc .;

⑥ Metabolic diseases: such as diabetes and its complications;

⑦ Patellar joint diseases: such as osteoarthritis, femoral head necrosis, etc .;

⑧Reproductive system diseases: such as premature ovarian failure, endocrine disorders, etc.

⑨ Other fields, such as corneal stem cells for eye diseases, stem cell anti-aging, etc.

Creative Biolabs can provide phenotypic, genetic, and functional assessments of your MSC population of interest using various techniques and tools. We adapt to your requirements by providing either individual service modules or a fully comprehensive service package.

Introduction to effective methods for exosomes isolation

Exosomes are nano-scale vesicles secreted by cells. These microvesicles are usually about 30-150 nanometers in diameter and contain important cellular molecules such as proteins and RNA. Previous studies have shown that exosomes can be used as diagnostic markers for cancer, neurodegenerative disease, and kidney disease. In recent years, exosomes isolation technology has made significant progress and development.

  1. Ultracentrifugation

Ultracentrifugation is the most commonly used exosomal purification method. After removing dead cells and cell debris by low speed centrifugation, high-speed centrifugation is used to precipitate vesicle particles of the same size from soluble molecules such as free proteins and protein complexes purified. It is important that the exosomes be subsequently washed at least once with PBS or fresh growth medium to reduce free residual proteins therein. In addition, all centrifugation steps must be performed at 4 ° C to keep the proteases, DNase and RNases inactive.

Usually ultracentrifugation is also used in combination with a sucrose density gradient (its continuous distribution from low to high density) or a sucrose cushion (30% sucrose cushion), that is, centrifuged at 100,000-200,000 xg in a centrifuge (containing exosomes) In 120 minutes, the exosomes in the sample should be enriched in a sucrose density range of 1.13-1.19 g / mL.

Although this powerful method can obtain highly purified exosomes, there are some disadvantages. Indeed, the process of ultracentrifugation is time-consuming and labor-intensive, and requires a lot of raw materials. The biggest drawback is that repeated centrifugation operations are likely to cause damage to exosomal vesicles and reduce their quality, or soluble proteins in the sample may form aggregates and clumps with exosomes to cause contamination.

  1. Ultrafiltration centrifugation

Considering that exosomes are cystic bodies with a size of several tens of nanometers, which are larger than ordinary proteins, exosomes can also be separated according to their size, such as ultrafiltration and size exclusion chromatography (SEC).

Ultrafiltration is the selective separation of samples using ultrafiltration membranes with different retention molecular weight (MWCO). That is, the solvent, ie, some small molecular substances, is filtered to the other side of the membrane, while high relative molecular mass substances larger than the membrane pore size are retained On the ultrafiltration membrane, the purpose of separating exosomes is achieved.

This method is simple and efficient, and does not affect the biological activity of exosomes. It is the best method for studying exosomal RNA because it produces greater RNA production than ultrafiltration and precipitation methods. It is also possible to pass a nanofiltration concentrator. However, the main disadvantage of ultrafiltration is that exosomes may block the filter pores, resulting in shorter membrane life and lower separation efficiency.

Exosome membranes also adhere to each other, resulting in low separation yields and even erroneous test results. In addition, there is another interference that needs to be resolved in the method of separating exosomes based on the size of the exosomes, which is the existence of a large number of non-exosomal nanovesicles that are similar in size to the exosomes.

In SEC, the porous phase fixed in the column can also be selectively separated based on the molecular size using the principle of gravity flow. Small molecules can pass through the pores and cause later elution, while larger components (including exosomes) can be eluted early, bypassing the pores. This method can greatly maintain the integrity and biological activity of exosomes, and combine with differential centrifugation to obtain highly purified exosomes.

  1. PEG-base precipitation method

Polyethylene glycol (PEG, 8000 kDa) can competitively bind free water molecules, so that less soluble molecules or exosomes are precipitated from the solution. Earlier this method was used to collect virus from samples such as serum, and now it is also used to precipitate exosomes. Samples are usually incubated overnight at 4 ° C with PEG, and exosomes are then recovered by low-speed centrifugation or filtration.

However, this method also has some problems: for example, the purity and recovery of exosomes are low, false positives (more proteins or some polymers that are difficult to remove), and mechanical or chemical additives that damage the exosomes.

Alternatively, if you know the sugar chain composition of the exosomes, you can use lectins to enrich the exosomes. Lectin is a protein that binds to carbohydrates and can be centrifuged at low speed after agglutinating exosomes. In recent years, exosomes have been separated based on the principle of precipitation. Various commercial exosomal extraction kits have also been developed on the market. The operation is simple, and high-purity and high-recovery exosomes can be obtained without ultracentrifugation.

  1. Magnetic bead immunoassay

Exosomes are available because they are rich in protein and have many specific marker receptors on their surface, such as CD9, CD81, CD63, CD82, Hsp70, Ras-related protein Rab-5b, cytoskeleton protein actin, and TSG101. Anti-marker antibody-coated magnetic beads can be captured after incubation with exosomes.

Because the heterogeneity of exosomes is consistent with their origin, the abundance of these markers on different exosomes is also different. Therefore, you can capture different types of exosomes from a sample by using specific antibody combinations, and select these exosomes by immobilizing these antibodies on ELISA plates, magnetic or chromatography beads, or microfluidic devices.

Although immunoaffinity technology has the advantages of high specificity, high purity exosomes can be obtained without affecting the morphological integrity of exosomes, it is the preferred method for enriching and characterizing unique exosomes. However, this method is low in efficiency, and the biological activity of exosomal contents is easily affected by pH and salt concentration, which is not conducive to the downstream experiments.

  1. Phosphatidylserine affinity

This method combines PS (phosphatidylserine) with magnetic beads and uses the principle of affinity to capture PS outside exosomal vesicles. This method is similar to the immunomagnetic bead method, and the exosomes obtained are complete in morphology and highest in purity. Since no denaturant is used and the biological activity of exosomes is not affected, exosomes can be used for cell co-culture and in vivo injection. 2016.9 “Scientific Reports” magazine published the latest data of this method, showing that PS method can extract relatively high purity exosomes.

  1. Chromatography

The exosomes isolated by this method are uniform in size under electron microscopy, but require special equipment and are not widely used.

Exosome isolation is the first step for exosome characterization. The quality of exosome separation directly affects the subsequent researches of exosome qualitative and quantitative as well as applications in disease diagnosis and therapy. With the extensive experience in exosome isolation, Creative Biolabs provides a portfolio of exosome isolation products which can help you with the high-quality exosome isolation from many types of biofluids in an efficient, faster and cheaper way.

 

Is It Really Healthy to Take Vitamin E For a Long Time?

Vitamin E is a fat-soluble vitamin that was discovered as early as the 1920s. Vitamin E includes tocopherols and triene tocopherols, a total of 8 compounds. Alpha-tocopherol is the most widely distributed and most abundant form of vitamin E in nature. Tocopherol is a hydrolysis product of Vitamin E and is one of the most important antioxidants.

 

How does Vitamin E help the human body?

 

Vitamin E helps delay aging

 

Vitamin E is a strong oxidant and is not weaker than lycopene and astaxanthin. After entering the body, vitamin E can help fight free radical Oxylipin peroxidation, eliminate free radicals, and delay aging.

 

Vitamin E helps boost immunity

 

If vitamin E is lacking, it will reduce the body’s humoral immunity and cellular immunity, and increase the possibility of human diseases. Proper vitamin E supplementation will help to strengthen the body’s ability to resist disease and enhance its physique.

 

Vitamin E helps eliminate pigmentation

 

Pigmentation is caused by the deposition of lipofuscin in skin cells. Lipofuscin is the product of cells being oxidized by free radicals. This substance not only produces stains and hinders aesthetic appearance, but also deposits in the internal organs and brain cells, causing cardiovascular and cerebrovascular diseases, and endangering health. Vitamin E as a strong oxidant can eliminate these free radicals, help prevent the generation of pigmentation, and at the same time tenderly expand peripheral blood vessels, reduce blood viscosity, and prevent cardiovascular and cerebrovascular diseases. Vitamin E can stabilize the protein active structure of the cell membrane, promote the normal development of muscles and maintain the elasticity of the skin, so that the skin and the body remain active; Vitamin E entering the skin cells can directly help the skin fight against the damage of free radicals, ultraviolet rays and pollutants, preventing The skin loses its elasticity due to some chronic or hidden injuries until it ages. Because of these effects of vitamin E, it is believed that vitamin E helps beauty.

 

Vitamin E helps protect eyesight

 

Vitamin E can inhibit the lipid peroxide response in the lens of the eye, expand the peripheral blood vessels, improve blood circulation, and prevent the occurrence and development of myopia.

 

Vitamin E helps relieve stomach ulcers

 

The poor gastric mucosal resistance in patients with ulcer disease is related to the disturbance of fat peroxidation. Vitamin E can regulate fat oxidation and scavenge oxidative free radicals, while protecting cells from oxidant damage. At the same time, a large amount of vitamin E can promote the proliferation of capillaries and small blood vessels, improve the surrounding blood circulation, increase the supply of oxygen in the tissue, thereby creating good nutritional conditions for healing of the ulcer surface. In addition, it can still inhibit the growth of H. pylori and reduce the recurrence rate of ulcer disease after healing.

 

Vitamin E helps promote sex hormone secretion

 

Vitamin E can increase men’s sperm vitality and quantity; increase women’s estrogen concentration, improve fertility, and prevent miscarriage.

 

Is it okay to take a lot of vitamin E for a long time?

 

Vitamin E is found in edible oils, fruits, vegetables and grains. The recommended daily intake for adults is 8 to 10 IU. Vitamin E in the general diet can completely meet the needs of the human body. Therefore, the general population does not need to take vitamin E for a long time. Long-term use is not only unsafe, but also has side effects.

 

Taking large doses of vitamin E for a long time may cause various diseases. The more serious ones are:

 

Intake of low-dose vitamin E has anti-oxidant effect, but it may no longer have antioxidant activity when ingested in large doses. At this time, vitamin E becomes a pro-oxidant;

 

Thrombophlebitis or pulmonary embolism, or both, is due to the high dose of vitamin E that can cause platelet aggregation and formation, which may trigger the risk of stroke;

 

Headache, dizziness, dizziness, blurred vision, muscle weakness; skin cracking, cheilitis, angular cheilitis, urticaria;

Methods of de novo drug design by computer-aiding (part two)

  1. Molecular fragmentation

The molecular fragment approach is nicknamed the chemical number holding algorithm or the fragment joining algorithm, and was proposed by Qu et al. in 1991. The basic structure applied by this method is fragments. Each fragment is composed of a single functional group (such as a hydroxyl group, a phenyl group, or a benzene ring, etc.). It is divided into fragment connection methods and fragment growth methods according to the different connection and growth methods of each fragment.

(1) Fragment connection method (or linked-fragment approach): first, there must be a fragment library storing various fragments and a linker library of various linkers. Linkers are -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH = CH-, -C00-, -CONH-, -O-. During the operation, a network is first created in the receiving point region of the receptor. The surface properties of the receiving point are analyzed with probe atoms, such as hydrophobicity, fluorine bonds, static electricity, and van der Waals’ gravitation. Subregions that can accommodate a fragment, such as hydrogen-bonded donors, hydrogen-bonded acceptors, fat hydrophobic, aromatic hydrophobic, and electrostatic interaction zones. Then search the debris library to find the matching shape and quality of the debris. Then search the linker library, find the appropriate linker, connect the fragments of each sub-region, and you can get a complete molecule. A series of molecules produced is optimized by molecular mechanical calculations to select the best ones The structure is for further study.

The software of the fragment connection method includes CAVFAT, SPLICE. HOOK, NEWLEAD and PRO-LIGAND.

(2) Fragment growth (fragment build): similar to the atom build, but at the receptor receiving point according to the nature and shape of the requirements, using fragments instead of atoms to grow one by one to build molecules. The starting point for fragment generation can be the seed atom specified on the recipient point, or the core fragment. Core fragments are fragments that can be attached to the acceptor point and can be obtained from the fragment library, or a fragment of the ligand molecule is selected. Then, fragments are grown according to the amount of energy, and a complete molecule is finally obtained. From a series of structures obtained by optimization of molecular mechanics, a reasonable structure was selected for further research.

Since the fragmentation method uses chemically reasonable fragments as the basic unit for design, the obtained new molecules are easy to accept in structure, so this method has become the mainstream of new drug design.

There are many design software for the fragment growth method, such as LUDI, LEAPFROG, SPROUT, CLIX, GROW, SPLICE, GROMOL and GEMINI.

LUDI (Biosym), which distinguishes four types of interaction sites between receptors and ligands:

(I) Hydrogen-bonded donors and hydrogen-bond acceptors can find sites where hydrogen bonds can be formed; (2) Aliphatic lipophilic sites and aromatic lipophilic sites, which can find suitable positions for hydrophobic interactions.

After determining the site of action, fill in the site with structural debris. Use a certain distance as a search criterion to search for suitable receiving points in groups of 2, 3, and 4 groups. A suitable fragment was retrieved from the library and filled in. If the fragment overlaps with the recipient without Fan Lihua and is electrically rejected, only the distance is appropriate, the fragment can be accepted. “The fragments are connected to form a fragment that is not connected with a real bond, and it is enough to simply lead the compound in the form of one or more points connected, and then artificially interfere, design and optimize the structure of the compound to obtain the lead compound.

LEAPFROG (Tripos) is similar to LUDI, but it can quickly calculate the energy of a large number of candidate compounds and eliminate compounds with inappropriate energy. It has three main functions for post-processing: (1) Structural optimization (Optimize), The lead compound is optimized for structure; (2) Dream, a new lead compound is fictional; (3) Guide, which guides the user’s proposed structure optimization scheme.

4.Homologous protein modeling

Direct drug design must know the three-dimensional structure of the receptor. However, so far, the X aromatic number receptor protein only knows the sequence of amino acids, and its three-dimensional structure is unknown, and there is no method to predict the three-dimensional structure of a protein from the primary structure of the protein.

Homologous protein maintains structural guidance during evolution, that is, homology.

For example, aspartic protease and peniclllopepsin, rhizopuspcpsin, fungal enzymes such as pepsin and cndothiapcpsin, and mammalian enzymes such as porcine pepsin, have a certain degree Homology. From the comparison of their structural sequences, it is not difficult to see that there are structurally conserved regions (SCRs) and variable regions (VRs) different from known structures, which will occupy the same positions of the structurally conserved regions. Arranged to get the structural sequence diagram of the aspartic protease.

Homologous proteins have similar spatial folding patterns and similar biological functions. Because the spatial structure of proteins is more conservative than that of sequences in evolution, sometimes two proteins with a certain sequence difference are still homologous. Therefore, homology can be identified based on the similarity of the spatial structure between proteins. Using the homology of proteins, using homologous proteins with known three-dimensional structure as a template can be used to model homologous proteins. This method is called homologous model building method, comparative molecular simulation method (comparative molecular modeling), homology modeling, or protein homology. Although the homologous molecular method has some errors, it is still a very practical method to obtain the three-dimensional structure of homologous proteins.

Homologous molecules: A homologous protein with a known secondary structure is used as a template. The structure of the target protein with a known sequence and a three-dimensional structure is estimated and modeled.

The basic steps of homologous source protein modeling:

(1) Report the amino acid sequence of an unknown protein. Look for one or several homologous proteins in the protein database. Use as a template for estimating and modeling unknown target proteins

(2) Display and overlap the primary sequence of the target protein and the template protein;

(3) Find structurally conserved regions of the template through structural comparison and sequence analysis;

(4) Find the unknown target molecule and the template of the known structure and divide the common secondary structure region to construct the local main chain structure of the target molecule:

(5) Connect the secondary structure fragments in the target molecule according to the known structure;

Molecular dynamics and molecular dynamics methods were used to optimize the initial model of the model to obtain the structure of the unknown protein.

Software for homologous protein modeling is available from many companies, such as Homology from MSI, Consensus from Biosym, Composer from Tripos, and Insight II, Modeler.

Creative Biolabs provides professional antibody modeling assessment services for antibody modeling. With the company’s most devoted scientists, it is confident to deliver researchers the comprehensive and professional assessment to meet various project development.

Antibody Homology Model

The three-dimensional (3D) structure of antibodies offers an understanding of their function and evolution, and assists in drug design and optimization. When an experimental structure is unavailable, the antibody’s 3D structure is usually obtained through comparative modeling, also known as homology modeling, as well as via de novo computational methods. Commonly, there are four steps to construct a homology model: template selection, template–target sequence alignment, model building, and model evaluation. Besides, many sequence alignment tools and protein structure databases are available to meet the task, such as the Protein Data Bank (PDB), which has more than 1000 crystal structures of antibody fragments (Fab or Fv).

The latest progress of CD20 CAR-T in the treatment of B-cell acute lymphocytic leukemia

According to previous data, CD19 was a hot CAR-T treatment target, and has achieved great success in relapsed and refractory B-cell malignant blood diseases, and there are already two commercial products for the treatment of relapse fefractory B-cell acute lymphocytic leukemia (B-ALL) and relapsed refractory non-Hodgkin’s lymphoma (NHL).

But not all patients benefit from CD19 CAR-T therapy. For example, most clinical data reports indicate that CD19 CAR-T has an objective response rate (ORR) of 80% and a complete response rate (CR) of approximately 50% in patients with relapsed and refractory NHL, and nearly 20% of non-responses and more than half of the patients fail to achieve sustained remission.

Why can’t some non-Hodgkin’s lymphoma patients benefit from it? Why is it?

Because the CD19 antigen is lost on the cancer cells of these patients, for these patients, CD19 CAR-T no longer has the ability to target and attack cancer cells, which in turn can lead to the recurrence of cancer.

How to solve this problem?

CAR-T therapy targeting CD20 may be an alternative solution.

CD20 is a human B lymphocyte restriction differentiation antigen, encoded by the MS4A1 gene (located at 11q12). This antigen is a hydrophobic 4-pass transmembrane protein with a molecular weight of approximately 35 kD, Leukocyte surface antigen Leu-16, transmembrane 4 domain subfamily A member 1 and so on. This protein function may be involved in regulating B cell activation and proliferation, and may function as a calcium ion channel.

CD20 antigen is mainly present on pre-B and mature B lymphocytes, and is expressed on most B-cell non-Hodgkin lymphoma cells, but not on stem cells, pro-B cells, normal plasma cells, or other normal organizations. Plasma cells naive and stimulated plasma cells may express CD20.

According to the expression characteristics of CD20 in the development stage of B lymphocytes, it has been selected as one of the targets for the treatment of B-cell lymphoma and leukemia, and many antibody drugs have been successfully developed based on this target.

Research progress of CD20 CAR-T therapy

  1. In China

As early as the end of 2012, the Molecular Immunology Department / Biotherapy Ward of the General Hospital of the Chinese People’s Liberation Army launched a clinical trial of CD20 as a target for CAR-T cell therapy for relapsed and refractory diffuse large B-cell lymphoma (DLBCL). The study, completed in 2014 and first reported the results of a phase I clinical study in 7 patients, showed that CD20 CAR-T cells combined with a tumor-reducing pretreatment regimen can prolong tumor regression (Wang Y, et al. ClinImmunol. 2014). At the same time, the team also applied to the State Intellectual Property Office for a CD20 CAR-NKT patent (engineered CD20-targeted NKT cells and its preparation method and application, patent application number: 201410062069.7).

At the beginning of 2015, Sibeman chose to cooperate with Han Weidong, director of the Molecular Immunology Laboratory of the Life Sciences Institute of the General Hospital of the PLA, to help Professor Han Weidong develop CD20 as a representative by leveraging his successful experience in translational medicine in the biomedical field and sufficient R & D funding advantages.

Based on the phase I clinical research, Han Weidong’s team launched a phase IIa clinical study of CD20 CAR-T in the treatment of relapsed and refractory NHL. The results of related studies in 11 patients were included and the results were published in “Signal Transduction and Targeted Therapy” in October 2016. Six of the 11 patients were evaluated as CR after CAR-T infusion (1 of whom was transferred from Phase 1 to Phase IIa and continued CD20 CAR-T alone), 3 were PR, and 2 were stable (SD ), 2 patients (1 PR, 1 SD) also received CR after local radiotherapy in the later stage.

In early October 2017, the Han Weidong team published a research report again in the “Signal Transduction and Targeted Therapy”. Based on a retrospective review of the clinical outcomes of 16 patients who can be evaluated after the test, the article highlights that 8 patients achieved CR after CAR-T infusion (or combined local radiotherapy), with the exception of 3 patients who had recurrence. As of the end of July 2017, 5 patients were still in the state of continuous CR (1 from the stage I subjects and 4 from the stage IIa subjects), of which 1 patient continued the CR stage and it has lasted 57 months, 3 cases exceeded 40 months, and 1 case exceeded 20 months. At the same time, in July 2017, the NCI in the United States reported the long-term follow-up results of CD19-CAR-T in the treatment of NHL in Molecular Therapy, showing 5 of 7 patients with CR, 4 of whom had a continuous CR period of 56, 51, 44, 38 months.

These two studies show that CAR-T treatment can not only achieve short-term efficacy in patients with relapsed and refractory NHL, but also can obtain long-term CR efficacy in most patients who obtain CR after CAR-T. It also indicates the long-term effectiveness of CAR-T therapy for CD20 in patients with relapsed and refractory NHL.

In terms of long-term safety, Han Weidong’s team also observed that patients with continuous CR after CAR-T treatment are often accompanied by longer-term B cell deficiency or low, and low immunoglobulinemia. Of the 5 long-term CR patients, except for one patient who developed grade 3 shingles infection in July after CAR-T treatment, the remaining patients can effectively prevent the occurrence of grade 3 infectious diseases through regular supplementation of gamma globulin .

  1. In US

In September 2017, Fred Hutch, a top cancer center, licensed a CD20 CAR-T therapy developed by Mustang Bio to the clinic as soon as possible.

Phase I / II clinical trials with partial support from Mustang Bio will be led by Dr. Mazyar Shadman, Clinical Research, Fred Hutch. The trial will recruit approximately 30 patients with relapsed or refractory B-cell non-Hodgkin’s lymphoma (B-NHL). Eligible patients will first undergo a biopsy to ensure that their tumors have a CD20 marker. The researchers expressed hope that as shown in preclinical studies, CD20 CAR-T therapy could even be more effective than CD19 CAR-T.

What is bioactive glass?

Glass is used frequently in daily life, and it is quite impossible to get rid of glass. Glass is stable, proof to acid and alkali, and hard and durable. It is one of the raw materials required for most important equipment. In this article, we mainly focus on bioglass and make a comparison between the two.

Bioactive glass (BAG) has attracted wide attention in the fields of chemical materials and medicine due to its good biocompatibility, bioactivity, bone conductivity and degradability. At present, bioactive glass has been successfully used in the fields of bone injury and the treatment and repair of dental diseases. In this article, the composition and structure of biologically active glass, its preparation method, active mechanism and application in the field of bone repair and dentistry were described, and its development prospect was also prospected.

  1. Doesbioglass equal to bioactive glass?

The answer is yes. Bioglass (BG) or bioactive glass is a glass that can achieve specific biological and physiological functions. Bioglass is implanted into the bone defect site of the human body, and it can be directly combined with bone tissue to repair bone tissue and restore its function. Bioglass is invented by L.L. Henky from Florida University in 1969. Its main ingredients are approximately 45% Na2O, 25% CaO and 25% SiO2, and approximately 5% P2O5. If a small amount of other ingredients are added, such as K2O, MgO, CaF2, B2O3, etc., can obtain a series of practical biological glass. Using this glass to make human bones is much better than some metals.

  1. The characteristics of bioactive glass

BAG is a silicate glass composed of basic components such as SiO2, Na2O, CaO and P2O5. The significant feature of bioactive glass and glass ceramics is that the surface conditions change dynamically with time after implantation into the human body, and a bioactive hydroxyapatite (HCA) layer is formed on the surface, which provides a bonding interface for the tissue. Most bioactive glass is a Class A bioactive material, which has both osteoproductive and osteoconductive effects, and has good binding with bone and soft tissues.

  1. The application of bioactive glass

BAG is considered to be a good biological material applicable in the field of repair. The use of this repair material is not only extremely extensive, but also has irreplaceable magical effects on professional products, such as skin care, whitening and wrinkling, burns, oral ulcers, gastrointestinal ulcers, skin ulcers, and fungal killing , bone repair, bonding of soft tissue and bone tissue, etc. It is believed that its appearance will make an outstanding contribution to human health. In addition, BAG also has a fast surface response; the amorphous two-dimensional structure makes the strength and fracture toughness low; the elastic modulus (30-35MPa) is low, close to the cortical bone; and machinable bioglass has good processing performance.

  1. The prospects of  bioglass

BG, as a special composition and structure of glass, has good bone conductivity, biological activity and other biological properties. It has good development prospects. However, its performance is far from meeting the requirements of ideal bone graft materials, and people need to further optimize its component structure, improve performance, and expand its use. For example, the component structure of BG can be adjusted through the adjustment of components, the selection of precursors, and the optimization of the preparation process to improve its mechanical strength, degradation rate, or give it special functions. By combining with other modern preparation technologies, such as electricity Spinning, 3D printing technology, etc., preparing BGs with various morphologies and different spatial structures could inspire new uses.

Terms

Material

Materials are those that humans use to make goods, devices, components, machines, or other products, which include natural material and artificial material. Materials are substances, but not all substances can be called materials. Materials such as fuels and chemical materials, industrial chemicals, food and drugs are generally not considered as materials. However, this definition is not so strict. For example, solid rocket propellants are generally called “energetic materials” because they are part of artillery or rockets.

A living thing

A living thing with kinetic energy is also a collection of objects, while an individual creature refers to a living body, as opposed to a non-living body. Its elements include: under natural conditions, living objects with survivability and reproduction ability generated by chemical reactions and living offspring produced by it (or them) through reproduction, can respond to external stimuli accordingly , can be interdependent with the external environment and promote each other. In addition, it can excrete unnecessary substances in the body, and has the characteristics of inheritance and mutation.

 

What factors determine the treatment effect of MSC (Part One)

The clinical research of MSC has been carried out for many years, but various treatment results have been obtained. Why so? What kind of factors affect the results?  Here in this article, we summarized several possible reasons based on study results and clinical practice.

Mesenchymal stem cells (MSCs) are one of the stars in clinical applications. There have been many reports of mesenchymal stem cells trying to treat a variety of diseases. It can be used to “treat” a variety of diseases, but it does not mean that MSC is the best or only choice for these diseases, nor does it mean that MSC can cure these diseases. It can only be said that researchers have tried MSC to treat these diseases. And some get pretty good results.

There are many factors that affect the efficacy of MSC. This article mainly focus on cell quality, injection route, optimal dose, and timing of treatment. Of course there are other factors, such as the type of disease, the course of the disease, the medical technology implemented by the doctor, and the overall strength of the hospital.

  1. Cell quality

There are some differences in the quality of the same drug produced by different manufacturers. The same situation exists for different stem cell companies.

Let’s first define what is “cell quality”?

Cell quality refers to the biological potency of a unit cell or a single cell; the higher the potency, the better the cell quality.

Efficacy includes two factors, strength and effectiveness, that is, the effect achieved at the same strength (generally refers to concentration). Therefore, when we talk about efficacy, we should be specific. The concept of efficacy is widely used in the biological world, including pesticides and drugs. This concept is also applied to the field of stem cells.

Therefore, when we talk about the cell quality or biological efficacy of MSC, specific indications are specific. We should talk about how effective this is in a range of indications. For example, MSC has both the function of immunosuppression and the promotion of vascular regeneration, and two different types of diseases are treated separately, then there are two indicators of biological efficacy.

However, the basic and clinical research of MSC has not yet entered this subdivided field. Here we will talk about the cell quality and what factors are relevant.

There are some parameters that can reflect the quality of mesenchymal stem cells, such as cell viability, donor characteristics, clone formation ability, cell size, immunosuppressive ability, and cytokine secretion. Here is a brief introduction to cell viability and donor characteristics.

  1. A. Cell viability

It refers to the alive number of MSCs before MSC injection. It is rare in the literature to explicitly mention the cell viability of MSCs. The cell viability used in different clinical studies is different, including > 80%,> 85%, 88.2 ± 6.1%, 90% -97%,> 92%,> 95%. There are also cases > 70%.

Cell preparations with a viability of 90% or more and with only 70% have a large difference in treatment effect under the same disease conditions. After all, MSC needs to be alive, and it needs to be cleared from the lungs in order to play a good therapeutic role. So, cell viability is really important!

  1. Donor characteristics

Donor characteristics include “donor age” and “donor physical condition”.

(1) Donor age

Donor age is an important factor because MSCs from young donors appear to have greater viability, proliferation potential, and antioxidant capacity, while older adult-sourced MSCs have lower proliferation capacity. In terms of age, umbilical cord, umbilical cord blood, and placenta should have the most advantages, followed by deciduous teeth and pulp, while bone marrow and fat are relatively older.

The older the age, the fewer stem cells are in the bone marrow. At birth, 1 of the 10,000 mononuclear cells in the bone marrow is MSC. At 30 years of age, the number of MSCs is reduced to 250,000 mononuclear cells in the bone marrow. By the age of 80, the number of MSCs will be even smaller, and only 2,000,000 MSCs will have one MSC.

Gender may affect some functions of the MSC. For example, studies have shown that female-derived MSCs express higher levels of IFN-γR1 and IL-6β, and thus have stronger immunosuppressive capabilities.

So what are the functional differences between MSCs from different sources? MSCs from different tissues have certain differences, which are mainly reflected in the proliferation rate, secreted cytokine profile, and immunoregulatory ability of MSCs.

(2) Physical condition of the donor

Many studies have proved that the disease also affects the function of autologous mesenchymal stem cells, especially in some patients with autoimmune diseases, the MSCs of their own bone marrow have abnormal functions, including slower proliferation rate, reduced colony formation ability, and decreased immune suppression changes, such as the decrease in the amount of secreted growth factors, make the patient’s own bone marrow MSCs unsuitable for the treatment of their own diseases.

Theoretically, there may be some kind of congenital genetic mutation that causes the function of MSC to be defective, so MSCs derived from umbilical cord, cord blood, and placenta at the time of birth are not suitable for autologous treatment. However, no such article has been reported so far, but this possibility exists theoretically.

Do all patients have pathological changes of bone marrow MSCs? Not all. It is estimated that the function of bone marrow MSCs in patients may not be damaged in the early stage of the disease.

To be continued in Part Two…