Peg nhs

The applied hydrogel system was formed based on fibrin conjugates with PEG. To prepare the conjugates, homobifunctional PEG—O,O′-bis[2-(N-succinimidyl-succinylamino)-ethyl]polyethylene glycol (PEG-NHS; Sigma-Aldrich, Germany) was used. PEG-NHS was dissolved in a sterile phosphate buffer in the concentration of 1.5 mg/mL. The prepared PEG-NHS solution was added to a solution of fibrinogen (25 mg/mL) in the molar ratio of 5:1 (PEG-NHS: fibrinogen). The reaction was carried out for no less than 2 h at a temperature of 37 °C. The conjugates synthetized were routinely characterized using FTIR spectroscopy and had spectra similar to the typical ones previously published in our papers (the increased band at 1100 cm⁻¹ which corresponded to the PEG-derived (C–O) units’ insertion) [39 (link), 40 (link), 42 (link)]. The gel was formed upon addition of an equal volume of thrombin with the concentration of 5 U/mL to the modified fibrinogen. Prior to the surgical intervention, the modified fibrinogen either with the added cell suspension or without the cell suspension and thrombin were kept in sterile single use 1.5 mL plastic tubes.

Human blood plasma cryoprecipitate obtained from the GBUZ NO blood center (Nizhny Novgorod, Russia) was used as a source of fibrinogen. The protein was PEGylated (PEG-NHS; Sigma-Aldrich, Darmstadt, Germany). Then a solution of 2% collagen was added—either CC isolated from cod skin [54 ] or BA—bovine collagen (Sigma-Aldrich, Germany). Phosphate buffer (PBS) was added to the resulting mixture at a 7:1 ratio. To polymerize the mixture, a solution of human thrombin (80 U/mL; Sigma-Aldrich, Germany) in 1% CaCL₂ solution was introduced into it. Studies with the scaffolds were carried out 24 h after their formation. For this, the newly formed scaffolds were flushed with 5 mL of PBS and incubated in a CO₂ incubator [45 ,75 (link)].

For the formation of scaffolds, blood plasma cryoprecipitate obtained from healthy donors was used [27 (link),29 ]. The plasma cryoprecipitate was PEGylated using PEG-NHS (Sigma-Aldrich, Darmstadt, Germany). Then a 2% collagen solution was added (PH = 7.4). In our research, type I collagen isolated from cod skins was used [30 ,31 (link)]. The resulting composite was injected with a cell suspension in phosphate buffer. The concentration of cells per 1 mL of the composite was 1.2 × 10⁵. The formation of scaffolds took place under the conditions of an enzymatic hydrolysis reaction. The composite was injected with a thrombin-calcium mixture: 80 IU/mL of human thrombin (NPO RENAM, Moscow, Russia) in 1% CaCL₂ solution. Scaffolds were formed within 20 min at a temperature of 22 to 25 °C. The scaffolds were then transferred to a plastic Petri dish. The scaffolds were cultured in complete growth medium. The cultivation was carried out in a CO₂ incubator at 37 °C, with a humidified atmosphere, and 5% CO₂ content.
The resulting scaffolds were dimensionally stable and transparent (Figure 1A). The internal structure of the scaffolds was characterized by their heterogeneous porosity (Figure 1B).

To form cell-free scaffolds (CFSs) and cell-containing scaffolds (CCSs), a composite based on human blood plasma cryoprecipitate was used [23 ]. To PEGylate the cryoprecipitate component, PEG-NHS (Sigma-Aldrich, Germany) was injected. A 2% collagen solution (marine collagen isolated from cod skins) was added to the PEGylated cryoprecipitate, pH = 7.2 to 7.4 [24 ]. In those studies, during CCS formation, an MSC in PBS suspension was introduced into the composite. As the CCS was intended for future use in humans, human adipose tissue MSCs were used. In our study of CCSs, the human MSCs were substituted with rat adipose tissue MSCs according to the “homologous drug” strategy. The rat cell concentration was 1.2 × 10⁵ per 1 mL of composite. During the formation of the cell-free scaffolds, a phosphate buffer solution (PBS) was injected into the composite in a 7:1 ratio. To polymerize the composite, a thrombin-calcium mixture was added: 80 IU/mL thrombin (Sigma-Aldrich, Germany) in 1% CaCl₂ solution. The CCSs were cultured for 3 days in a complete growth medium under CO₂ incubator conditions.
Before the experiment, the CCSs and CFSs were washed three times with the phosphate buffer solution and then sliced using a template into equal 1 cm² fragments. A macroscopic image of the scaffold samples is shown in Figure 1.

Polyethylenimine (PEI, Sigma) was fully dissolved in KH₂PO₄/NaOH solution. Then Polyethylene glycol (PEG)-NHS (Sigma) was added and slowly stirred for 2 days 1.44 ml PEI/PEG solution was added into 25 ml HAuCl₄ aqueous solution (42 mM, Sigma) and slowly stirred for 1 day. Then centrifuged at 12000g for 10 min, discarded the supernatant, and washed with deionized water for three times. OX40 aptamer-PD-1 siRNA chimeras or PD-1 siRNA (synthesized by GenePharma) were added, stirred gently in dark for half an hour, centrifuged at 12000g for 10 min, and discarded the supernatant. After washing with deionized water for three times, aptamer-siRNA chimeras/PEI/PEG/AuNP nanoparticles were obtained.
10 mg aptamer-siRNA chimeras/PEI/PEG/AuNP nanoparticles were dissolved in 10 ml collagen solution (Sigma) and stirred slowly at 4°C for 2 h. Then 150 uL collagen solution was carefully drizzled onto the glass slide and then freeze-dried to form the monolayer membrane. Chimeras/PEI/PEG/AuNP/collagen monolayer membrane was put into heparin solution (1 mg/ml, Sigma). After 15 min, another monolayer membrane was gently put on the top of first monolayer membrane and cross-linked for 15 min. After 10 repetitions, collagen membranes were carefully washed with ionized water for three times. In the end, freeze drying was performed to obtain chimeras/PEI/PEG/AuNP/collagen membranes.