Biodegradable Cell Microcarriers Based on Chitosan/Polyester Graft-Copolymers

Demina T.S., Kuryanova A.S., Aksenova N.A., Shubnyy A.G., Popyrina T.N., Sokovikov Y.V., Istranova E.V., Ivanov P.L., Timashev P.S., Akopova T.A.

Graft-copolymers based on bioresorbable synthetic (oligo-/polylactide) and natural (chitosan and collagen/gelatin) components were synthesized through solid-state reactive co-extrusion and used for fabrication of fibrous non-woven mats via the electrospinning technique. The effect of the macromolecular features of the initial components on the copolymer characteristics was evaluated using FTIR-spectroscopy, differential scanning calorimetry and elemental analysis. Dynamic light scattering analysis showed that the copolymers have a tendency to form stable ultra-fine dispersions with a mean size of macromolecular aggregates of 150 nm within chlorinated solvents. The copolymer-containing non-woven fibrous mats were fabricated via an electrospinning procedure using chloroform as a solvent. An effect of the copolymer composition on the casting solution's viscosity, conductivity and surface tension was evaluated. Scanning electron microscopy showed that the obtained mats consist of randomly distributed fibers with a mean size of ∼5 μm and a more complex morphology than mats fabricated from neat polylactide. The proposed mechanochemical approach to obtain hybrid copolymeric compositions differs from typical liquid-phase methods in terms of high efficiency, simplicity and cleanness.

Chen X., Chen J., Tong X., Mei J., Chen Y., Mou X.

Microcarriers are 100- to 300-micron support matrices that permit the growth of adherent cells in bioreactor systems. They have a larger surface area to volume ratio in comparison to single cell monolayers, enabling cost-effective cell production and expansion. Microcarriers are composed of a solid matrix that must be separated from expanded cells during downstream processing stages. The detachment method is chosen on the basis of several factors like cell type, microcarrier surface chemistry, cell confluency and degree of aggregation. The development of microcarriers with a range of physiochemical properties permit controlled cell and protein associations that hold utility for novel therapeutics. In this review, we provide an overview of the recent advances in microcarrier cell culture technology. We also discuss its significance as an ex vivo research tool and the therapeutic potential of newly designed microcarrier systems in vivo.

Derakhti S., Safiabadi-Tali S.H., Amoabediny G., Sheikhpour M.

Achieving a high cell density of animal cells is a prerequisite for different medical applications such as cell therapy, tissue engineering, and vaccine production. Microcarrier-based cell culture has been proved to be a promising method to attain this purpose mainly due to providing a high surface area to volume ratio. Adhesion and harvesting of cells to and from microcarriers are two critical stages influencing final cell productivity and quality. Low attachment efficiency or non-uniform initial cell distribution onto microcarriers' surfaces lead to the waste of inoculum and achievement of cellular yields less than expected. In other side, inappropriate detachment procedure decreases cell recovery along with having adverse effects on cell viability and behavior. In this review, a comprehensive study on these crucial steps is presented. In the attachment section, cellular mechanisms involved in the attachment process are briefly discussed. Due to the key role of microcarrier surface characteristics in cell attachment and behavior, the chemistry and physical features of various microcarrier surfaces are studied in detail. Then, the influence of seeding conditions on cell attachment is reviewed. In the detachment section, chemical harvesting methods are described initially followed by mechanical detachment. Finally, thermo-responsive microcarriers are discussed in detail. At the end of each section, current challenges and future directions are highlighted.

Neto M.D., Oliveira M.B., Mano J.F.

For several decades microparticles have been exclusively and extensively explored as spherical drug delivery vehicles and large-scale cell expansion carriers. More recently, microparticulate structures gained interest in broader bioengineering fields, integrating myriad strategies that include bottom-up tissue engineering, 3D bioprinting, and the development of tissue/disease models. The concept of bulk spherical micrometric particles as adequate supports for cell cultivation has been challenged, and systems with finely tuned geometric designs and (bio)chemical/physical features are current key players in impacting technologies. Herein, we critically review the state of the art and future trends of biomaterial microparticles in contact with cells and tissues, excluding internalization studies, and with emphasis on innovative particle design and applications.

Demina T.S., Sevrin C., Kapchiekue C., Akopova T.A., Grandfils C.

Baakdhah T., van der Kooy D.

Blindness as a consequence of degenerative eye diseases (e.g., age-related macular degeneration and retinitis pigmentosa) is a major health problem and numbers are expected to increase by up to 50% by 2020. Unfortunately, adult mouse and human retinal stem cells (RSCs), unlike fish and amphibians, are quiescent in vivo and do not regenerate following disease or injury. To replace lost cells, we used microcarriers (MCs) in a suspension stirring bioreactor to help achieve numbers suitable for differentiation and transplantation. We achieved a significant 10-fold enrichment of RSC yield compared to conventional static culture techniques using a combination of FACTIII MCs and relative hypoxia (5%) inside the bioreactor. We found that hypoxia (5% O2 ) was associated with better RSC expansion across all platforms; and this can be attributed to hypoxia-induced increases in survival and/or symmetric division of stem cells. In the future, we will target the differentiation of RSCs and their progeny toward rod and cone photoreceptor phenotypes using FACTIII MCs inside bioreactors to expand their populations in order to produce the large numbers of cells needed for transplantation.

Tavassoli H., Alhosseini S.N., Tay A., Chan P.P., Weng Oh S.K., Warkiani M.E.

Human stem cells, including pluripotent, embryonic and mesenchymal, stem cells play pivotal roles in cell-based therapies. Over the past decades, various methods for expansion and differentiation of stem cells have been developed to satisfy the burgeoning clinical demands. One of the most widely endorsed technologies for producing large cell quantities is using microcarriers (MCs) in bioreactor culture systems. In this review, we focus on microcarriers properties that can manipulate the expansion and fate of stem cells. Here, we provide an overview of commercially available MCs and focus on novel stimulus responsive MCs controlled by temperature, pH and field changes. Different features of MCs including composition, surface coating, morphology, geometry/size, surface functionalization, charge and mechanical properties, and their cellular effects are also highlighted. We then conclude with current challenges and outlook on this promising technology.

Li L., Song K., Chen Y., Wang Y., Shi F., Nie Y., Liu T.

Nowadays, microcarriers are widely utilized in drug delivery, defect filling, and cell culture. Also, many researchers focus on the combination of synthetic and natural polymers and bioactive ceramics to prepare composite biomaterials for tissue engineering and regeneration. In this study, three kinds of microcarriers were prepared based on physical doping and surface modification, named Poly (l-lactic) acid (PLLA), PLLA/nanohydroxyapatite (PLLA/nHA), and PLLA/nHA/Chitosan (PLLA/nHA/Ch). The physicochemical properties of the microcarriers and their functional performances in MC3T3-E1 cell culture were compared. Statistical results showed that the average diameter of PLLA microcarriers was 291.9 ± 30.7 μm, and that of PLLA/nHA and PLLA/nHA/Ch microcarriers decreased to 275.7 ± 30.6 μm and 269.4 ± 26.3 μm, respectively. The surface roughness and protein adsorption of microcarriers were enhanced with the doping of nHA and coating of chitosan. The cell-carrier cultivation stated that the PLLA/nHA microcarriers had the greatest proliferation-promoting effect, while the PLLA/nHA/Ch microcarriers performed the strongest attachment with MC3T3-E1 cells. Besides, the cells on the PLLA/nHA/Ch microcarriers exhibited optimal osteogenic expression. Generally, chitosan was found to improve microcarriers with superior characteristics in cell adhesion and differentiation, and nanohydroxyapatite was beneficial for microcarriers regarding sphericity and cell proliferation. Overall, the modified microcarriers may be considered as a promising tool for bone tissue engineering.

Bee S., Hamid Z.A., Mariatti M., Yahaya B.H., Lim K., Bee S., Sin L.T.

Shekaran A., Lam A., Sim E., Jialing L., Jian L., Wen J.T., Chan J.K., Choolani M., Reuveny S., Birch W., Oh S.

Human mesenchymal stromal cells or marrow stromal cells (MSCs) are of great interest for bone healing due to their multi-potency and trophic effects. However, traditional MSC expansion methods using 2-dimensional monolayer (MNL) flasks or cell stacks are limited by labor-intensive handling, lack of scalability, the need for enzymatic cell harvesting and the need for attachment to a scaffold before in vivo delivery. Here, we present a biodegradable microcarrier and MSC bioprocessing system that may overcome the abovementioned challenges.We cultured human early MSCs (heMSCs) on biodegradable polycaprolactone microcarriers (PCL MCs) coated with extracellular matrix (ECM) and evaluated the in vitro osteogenic differentiation and in vivo bone formation capacity of ECM-coated PCL MC-bound heMSCs compared with conventional MNL-cultured cells.We found that heMSCs proliferate well on PCL MCs coated with a fibronectin, poly-l-lysine, and fibronectin (FN+PLL+FN) coating (cPCL MCs). During in vitro osteogenic induction, heMSCs cultured on cPCL MCs displayed a 68% increase in specific calcium deposition compared with cultures on MNL. In a mouse ectopic mineralization model, bone mass was equivalent for MNL-expanded and cPCL MC-bound heMSC implants but higher in both cases when compared with cell-free cPCL MC implants at 16 weeks post-implantation. In summary, compared with MNL cultures, biodegradable MC MSC cultures provide the benefits of large-scale expansion of cells and can be delivered in vivo, thereby eliminating the need for cell harvesting and use of scaffolds for cell delivery. These results highlight the promise of delivering heMSCs cultured on cPCL MCs for bone applications.

Zhang Z., Eyster T.W., Ma P.X.

Biodegradable polymer microspheres have emerged as cell carriers for the regeneration and repair of irregularly shaped tissue defects due to their injectability, controllable biodegradability and capacity for drug incorporation and release. Notably, recent advances in nanotechnology allowed the manipulation of the physical and chemical properties of the microspheres at the nanoscale, creating nanostructured microspheres mimicking the composition and/or structure of natural extracellular matrix. These nanostructured microspheres, including nanocomposite microspheres and nanofibrous microspheres, have been employed as cell carriers for tissue regeneration. They enhance cell attachment and proliferation, promote positive cell-carrier interactions and facilitate stem cell differentiation for target tissue regeneration. This review highlights the recent advances in nanostructured microspheres that are employed as injectable, biomimetic and cell-instructive cell carriers.

Demina T.S., Akopova T.A., Vladimirov L.V., Zelenetskii A.N., Markvicheva E.A., Grandfils C.

Amphiphilic chitosan-g-poly(d,l-lactide) copolymers have been manufactured via solid-state mechanochemical copolymerization and tailored to design polyester-based microspheres for tissue engineering. A single-step solid-state reactive blending (SSRB) using low-temperature co-extrusion has been used to prepare these copolymers. These materials have been valorized to stabilize microspheres processed by an oil/water emulsion evaporation technique. Introduction of the copolymers either in water or in the oil phase of the emulsion allowed to replace a non-degradable emulsifier typically used for microparticle preparation. To enhance cell adhesion, these copolymers were also tailored to bring amino-saccharide positively charged segments to the microbead surface. Size distribution, surface morphology, and total microparticle yield have been studied and optimized as a function of the copolymer composition.

Yang L., Zhang J., He J., Zhang J., Gan Z.

Porous microspheres of cellulose-graft-poly(L-lactide) (cellulose-g-PLLA) copolymers were fabricated by a water/oil/water (W/O/W) emulsion evaporation method. Their morphology, hydrophilicity and amount of hydroxyl group content (–OH content) on the surface were adjustable with the change of molar substitution of PLLA (MSPLLA). After a hydrolysis post-processing, surface physical properties of microspheres were regulated further. The influence of physical properties of microspheres on cell cultivation was investigated by setting hepatocellular liver carcinoma cell line (HepG-2) as the example. Compared with the cells on PLLA microspheres, HepG-2 cells with more pseudopods spread well on the surface of cellulose-g-PLLA microspheres. Particularly, the cellulose-g-PLLA microspheres with MSPLLA of 11.5 were the most appropriate candidate for cell adhesion and proliferation. Therefore, a moderate PLLA content of cellulose-g-PLLA copolymers was beneficial to the cell cultivation. This work indicated that combining cellulose with PLLA was an available route to developing cellulose-based materials for cell cultivation.

Ortiz M., Rosales-Ibáñez R., Pozos-Guillén A., De Bien C., Toye D., Flores H., Grandfils C.

Fiber scaffolds are attractive materials for mimicking, within a 3D in vitro system, any living environment in which animal cells can adhere and proliferate. In three dimensions, cells have the ability to communicate and organize into complex architectures similar to those found in their natural environments. The aim of this study was to evaluate, in terms of cell reactivity, a new in vitro cell model: dental pulp stem cells (DPSCs) in a 3D polymeric textile. Scaffolds were knitted from polyglycolic acid (PGA) or polydioxanone (PDO) fibers differing in surface roughness. To promote cell adhesion, these hydrophobic fabrics were also functionalized with either chitosan or the peptide arginine-glycine-aspartic acid (RGD). Cell behavior was examined 1, 10, and 21 days post-seeding with a LIVE/DEAD® Kit. Confocal laser scanning microscopy (CLSM) highlighted the biocompatibility of these materials (cell survival rate: 94% to 100%). Fiber roughness was found to influence cell adhesion and viability significantly and favorably. A clear benefit of polymeric textile functionalization with chitosan or RGD was demonstrated in terms of cell adhesion and viability. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 785-794, 2017.

Faia-Torres A.B., Charnley M., Goren T., Guimond-Lischer S., Rottmar M., Maniura-Weber K., Spencer N.D., Reis R.L., Textor M., Neves N.M.

The use of biomaterials to direct osteogenic differentiation of human mesenchymal stem cells (hMSCs) in the absence of osteogenic supplements is thought to be part of the next generation of orthopedic implants. We previously engineered surface-roughness gradients of average roughness (Ra) varying from the sub-micron to the micrometer range (∼0.5-4.7 μm), and mean distance between peaks (RSm) gradually varying from ∼214 μm to 33 μm. Here we have screened the ability of such surface-gradients of polycaprolactone to influence the expression of alkaline phosphatase (ALP), collagen type 1 (COL1) and mineralization by hMSCs cultured in dexamethasone (Dex)-deprived osteogenic induction medium (OIM) and in basal growth medium (BGM). Ra∼1.53 μm/RSm∼79 μm in Dex-deprived OI medium, and Ra∼0.93 μm/RSm∼135 μm in BGM consistently showed higher effectiveness at supporting the expression of the osteogenic markers ALP, COL1 and mineralization, compared to the tissue culture polystyrene (TCP) control in complete OIM. The superior effectiveness of specific surface-roughness revealed that this strategy may be used as a compelling alternative to soluble osteogenic inducers in orthopedic applications featuring the clinically relevant biodegradable polymer polycaprolactone.Biodegradable polymers, such as polycaprolactone (PCL), are promising materials in the field of tissue engineering and regenerative medicine, which aims at creating viable options to replace permanent orthopedic implants. The material, cells, and growth-stimulating factors are often referred to as the key components of engineered tissues. In this article, we studied the hypothesis of specific surface modification of PCL being capable of inducing mesenchymal stem cell differentiation in bone cells in the absence of cell-differentiating factors. The systematic investigation of the linearly varying surface-roughness gradient showed that an average PCL roughness of 0.93 μm alone can serve as a compelling alternative to soluble osteogenic inducers in orthopedic applications featuring the clinically relevant biodegradable polymer polycaprolactone.

Popyrina T.N., Minaeva E.D., Emelyanov K.V., Ilina E.B., Akopova T.А., Minaev N.V., Demina T.S.

AbstractModification of the chitosan chemical structure by grafting hydrophobic fragments makes it possible to synthesize derivatives with amphiphilic properties. These derivatives were used as emulsifiers in the aqueous phase during the production of polylactide microparticles via the oil/water solvent evaporation technique. The application of the derivatives allows to achieve a high yield of the microparticles and to provide a hydrophilic coating on the surface of polylactide microparticle. The chitosan‐coated polylactide microparticles were analyzed in terms of mean size, size distribution, surface, and internal morphology. The ability of the obtained microparticles to be used as a starting component for the fabrication of 3D materials through the surface‐selective laser sintering was evaluated, and the optimal parameters for the process have been determined.

Kong R., Chen J., Zhao F., Li Y., Yang H., Zheng Y., He W.

Handral H.K., Wyrobnik T.A., Lam A.T.

Microcarriers (MCs) are adaptable therapeutic instruments that may be adjusted to specific therapeutic uses, making them an appealing alternative for regenerative medicine and drug delivery. MCs can be employed to expand therapeutic cells. MCs can be used as scaffolds for tissue engineering, as well as providing a 3D milieu that replicates the original extracellular matrix, facilitating cell proliferation and differentiation. Drugs, peptides, and other therapeutic compounds can be carried by MCs. The surface of the MCs can be altered, to improve medication loading and release, and to target specific tissues or cells. Allogeneic cell therapies in clinical trials require enormous volumes of stem cells, to assure adequate coverage for several recruitment locations, eliminate batch to batch variability, and reduce production costs. Commercially available microcarriers necessitate additional harvesting steps to extract cells and dissociation reagents, which reduces cell yield and quality. To circumvent such production challenges, biodegradable microcarriers have been developed. In this review, we have compiled key information relating to biodegradable MC platforms, for generating clinical-grade cells, that permit cell delivery at the target site without compromising quality or cell yields. Biodegradable MCs could also be employed as injectable scaffolds for defect filling, supplying biochemical signals for tissue repair and regeneration. Bioinks, coupled with biodegradable microcarriers with controlled rheological properties, might improve bioactive profiles, while also providing mechanical stability to 3D bioprinted tissue structures. Biodegradable materials used for microcarriers have the ability to solve in vitro disease modeling, and are advantageous to the biopharmaceutical drug industries, because they widen the spectrum of controllable biodegradation and may be employed in a variety of applications.

Minaev N.V., Minaeva S.A., Sherstneva A.A., Chernenok T.V., Sedova Y.K., Minaeva E.D., Yusupov V.I., Akopova T.A., Timashev P.S., Demina T.S.

Biodegradable polyester/hydroxyapatite microparticles are widely proposed as microcarriers for drug/cell delivery or scaffolds for bone tissue regeneration. The current research implements the surfactant-free approach for the fabrication of polyester-based microparticles filled with hydroxyapatite nanoparticles (nHA) via the oil/water Pickering emulsion solvent evaporation technique for the first time, to the best of our knowledge. The process of polyester microparticle fabrication using nHA for the oil/water interface stabilization was studied as a function of phase used for nHA addition, which allows the preparation of a range of microparticles either filled with nHA or having it as a shell over the polymeric core. The effect of processing conditions (polymer nature, polymer/nHA ratio, ultrasound treatment) on particles’ total yield, size distribution, surface and volume morphology, and chemical structure was analyzed using SEM, EDX, Raman spectroscopy, and mapping. Addition of nHA either within the aqueous or oil phase allowed the effective stabilization of the oil/water interface without additional molecular surfactants, giving rise to hybrid microparticles in which total yield, size distribution, and surface morphology depended on all studied processing conditions. Preliminary ultrasound treatment of any phase before the emulsification process led to a complex effect but did not affect the homogeneity of nHA distribution within the polymeric core of the hybrid microparticles.

Sherstneva A.A., Demina T.S., Monteiro A.P., Akopova T.A., Grandfils C., Ilangala A.B.

Tissue engineering and cell therapy are very attractive in terms of potential applications but remain quite challenging regarding the clinical aspects. Amongst the different strategies proposed to facilitate their implementation in clinical practices, biodegradable microparticles have shown promising outcomes with several advantages and potentialities. This critical review aims to establish a survey of the most relevant materials and processing techniques to prepare these micro vehicles. Special attention will be paid to their main potential applications, considering the regulatory constraints and the relative easiness to implement their production at an industrial level to better evaluate their application in clinical practices.

Kochetkova O.Y., Demina T.S., Antonova O.Y.

Several variants of hybrid polyelectrolyte microcapsules (hPEMC) were designed and produced by modifying in situ gelation methods and layer-by-layer (LbL) techniques. All of the hPEMC designs tested in the study demonstrated high efficiency of the model hydrophilic compound loading into the carrier cavity. In addition, the microcarriers were characterized by high efficiency of incorporating the model hydrophobic compound rhodamine B isothiocyanate (RBITC) into the hydrophobic layer consisting of poly-(d,l)-lactide-co-glycolide (PLGA), oligo-(l)-lactide (OLL), oligo-(d)-lactide (OLD) and chitosan/gelatin/poly-l-lactide copolymer (CGP). The obtained microcapsules exhibited high storage stability regardless of the composition and thickness of the polyelectrolyte shell. Study of the impact of hybrid polyelectrolyte microcapsules on viability of the adhesive L929 and suspension HL-60 cell lines revealed no apparent toxic effects of hPEMC of different architecture on live cells. Interaction of hPEMC with peritoneal macrophages for the course of 48 h resulted in partial deformation and degradation of microcapsules accompanied by release of the content of their hydrophilic (BSA–fluorescein isothiocyanate conjugate (BSA-FITC)) and hydrophobic (RBITC) layer. Our results demonstrate the functional efficiency of novel hybrid microcarriers and their potential for joint delivery of drugs with different physico-chemical properties in complex therapy.

Demina T.S., Popyrina T.N., Minaeva E.D., Dulyasova A.A., Minaeva S.A., Tilkin R., Yusupov V.I., Grandfils C., Akopova T.A., Minaev N.V., Timashev P.S.

Surface-selective laser sintering (SSLS) is a specific version of selective laser sintering, which allows one to fabricate 3D structures with well-defined architectonic via selective melting of microparticle surface without alteration of their core. This mode of laser sintering requires a well-designed surface properties of the microparticles to adsorb laser irradiation. Water was chosen as safer sensitive absorber of laser radiation with a wavelength of 1.9 μm, i.e. one with low absorption coefficient by polymeric core. Biodegradable polylactide microparticles were fabricated via oil/water emulsion solvent evaporation technique using tailored-made chitosan-based macromolecules, which provided the effective interface stabilization during the microparticle fabrication and well balanced microparticle surface hydrophilicity to adsorb water. Especially build SSLS set-up was designed in order to monitor the effectiveness of the 3D scaffold fabrication from the obtained microparticles and to adapt the optimal laser radiation parameters with a wavelength of 1.9 μm (e.g. speed, line density, power).

Demina T.S., Akopova T.A., Zelenetsky A.N.

Abstract The transition to green chemistry and biodegradable polymers is a logical stage in the development of modern chemical science and technology. In the framework of this review, the advantages, disadvantages, and potential of biodegradable polymers of synthetic and natural origin are compared using the example of polylactide and chitosan as traditional representatives of these classes of polymers, and the possibilities of their combination via obtaining composite materials or copolymers are assessed. The mechanochemical approach to the synthesis of graft copolymers of chitosan with oligolactides/polylactides is considered in more detail.

Dabiri S.M., Samiei E., Shojaei S., Karperien L., Khun Jush B., Walsh T., Jahanshahi M., Hassanpour S., Hamdi D., Seyfoori A., Ahadian S., Khademhosseini A., Akbari M.

An effective treatment of human diseases using regenerative medicine and cell therapy approaches requires a large number of cells. Cultivation of cells on microcarriers is a promising approach due to the high surface-to-volume ratios that these microcarriers offer. Here, multifunctional temperature-responsive microcarriers (cytoGel) made of an interpenetrating hydrogel network composed of poly(N-isopropylacrylamide) (PNIPAM), poly(ethylene glycol) diacrylate (PEGDA), and gelatin methacryloyl (GelMA) are developed. A flow-focusing microfluidic chip is used to produce microcarriers with diameters in the range of 100-300 μm and uniform size distribution (polydispersity index of ≈0.08). The mechanical properties and cells adhesion properties of cytoGel are adjusted by changing the composition hydrogel composition. Notably, GelMA regulates the temperature response and enhances microcarrier stiffness. Human-derived glioma cells (U87) are grown on cytoGel in static and dynamic culture conditions with cell viabilities greater than 90%. Enzyme-free cell detachment is achieved at room temperature with up to 70% detachment efficiency. Controlled release of bioactive molecules from cytoGel is accomplished for over a week to showcase the potential use of microcarriers for localized delivery of growth factors to cell surfaces. These microcarriers hold great promise for the efficient expansion of cells for the industrial-scale culture of therapeutic cells.

Olarte-Paredes A., Salgado-Delgado A.M., García-Fuentes J.J., Salgado-Delgado R., Cedillo-Valverde G., López-Lara T., Hernández-Zaragoza J.B., Castaño V.M.

Akopova T.A., Demina T.S., Khavpachev M.A., Popyrina T.N., Grachev A.V., Ivanov P.L., Zelenetskii A.N.

Hydrophobic derivatives of polysaccharides possess an amphiphilic behavior and are widely used as rheological modifiers, selective sorbents, and stabilizers for compositions intended for various applications. In this work, we studied the mechanochemical reactions of chitosan alkylation when interacting with docosylglycidyl and hexadecylglycidyl ethers in the absence of solvents at shear deformation in a pilot twin-screw extruder. The chemical structure and physical properties of the obtained derivatives were characterized by elemental analysis, FT-IR spectroscopy, dynamic light scattering, scanning electron microscopy, and mechanical tests. According to calculations for products soluble in aqueous media, it was possible to introduce about 5–12 hydrophobic fragments per chitosan macromolecule with a degree of polymerization of 500–2000. The length of the carbon chain of the alkyl substituent significantly affects its reactivity under the chosen conditions of mechanochemical synthesis. It was shown that modification disturbs the packing ability of the macromolecules, resulting in an increase of plasticity and drop in the elastic modulus of the film made from the hydrophobically modified chitosan samples.