Thank you for visiting Nature.com.The browser version you are using has limited support for CSS.For the best experience, we recommend that you use an updated browser (or turn off compatibility mode in Internet Explorer).In the meantime, to ensure continued support, we will display the site without styles and JavaScript.
Insulin nanoparticles (NPs) with high loading content have found different applications in different dosage forms.This work aims to evaluate the effect of freeze-drying and spray-drying processes on the structure of insulin-loaded chitosan nanoparticles, with or without mannitol as a cryoprotectant.We also assessed the quality of these nanoparticles by redissolving them.Before dehydration, the particle size of the chitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles was optimized to be 318 nm, the PDI was 0.18, the encapsulation efficiency was 99.4%, and the loading was 25.01%.After reconstitution, all nanoparticles, except those produced by a freeze-drying method without the use of mannitol, maintained their spherical particle structure.Compared to mannitol-containing nanoparticles dehydrated by either spray, mannitol-free spray-dried nanoparticles also showed the smallest mean particle size (376 nm) and the highest loading content (25.02%) with similar encapsulation rate (98.7%) and PDI (0.20) by drying or freeze-drying techniques.The dried nanoparticles by spray drying without mannitol also resulted in the fastest release of insulin and the highest efficiency of cellular uptake.This work shows that spray drying can dehydrate insulin nanoparticles without the need for cryoprotectants compared to conventional freeze drying methods, creating greater loading capacity, lower additive requirements and operating costs significant advantage.
Since its discovery in 19221,2,3, insulin and its pharmaceutical preparations have saved the lives of patients with type 1 diabetes (T1DM) and type 2 diabetes (T1DM).However, due to its properties as a high molecular weight protein, insulin is easily aggregated, broken down by proteolytic enzymes, and eliminated by the first-pass effect.People diagnosed with type 1 diabetes need insulin injections for the rest of their lives.Many patients initially diagnosed with type 2 diabetes also require long-term insulin injections.Daily insulin injections are a serious source of daily pain and discomfort for these individuals, with negative effects on mental health.As a result, other forms of insulin administration that cause less discomfort, such as oral insulin administration, are being extensively studied5 as they have the potential to restore the quality of life of approximately 5 billion people with diabetes worldwide.
Nanoparticle technology has provided a significant advance in attempts to take oral insulin4,6,7.One that effectively encapsulates and protects insulin from degradation for targeted delivery to specific body sites.However, the use of nanoparticle formulations has several limitations, mainly due to stability issues of particle suspensions.Some aggregation may occur during storage, which reduces the bioavailability of insulin-loaded nanoparticles8.In addition, the chemical stability of the polymer matrix of nanoparticles and insulin must also be considered to ensure the stability of insulin nanoparticles (NPs).Currently, freeze-drying technology is the gold standard for creating stable NPs while preventing unwanted changes during storage9.
However, freeze-drying requires the addition of cryoprotectants to prevent the spherical structure of NPs from being affected by the mechanical stress of ice crystals.This significantly reduces the loading of insulin nanoparticles after lyophilization, as the cryoprotectant occupies most of the weight ratio.Therefore, the produced insulin NPs are often found to be unsuitable for the manufacture of dry powder formulations, such as oral tablets and oral films, due to the need for large amounts of dry nanoparticles to achieve the therapeutic window of insulin.
Spray drying is a well-known and inexpensive industrial-scale process for producing dry powders from liquid phases in the pharmaceutical industry10,11.Control over the particle formation process allows for proper encapsulation of several bioactive compounds 12, 13 .Furthermore, it has become an effective technique for the preparation of encapsulated proteins for oral administration.During spray drying, water evaporates very quickly, which helps keep the temperature of the particle core low11,14, enabling its application to encapsulate heat-sensitive components.Before spray drying, the coating material should be thoroughly homogenized with the solution containing the encapsulated ingredients11,14.Unlike freeze-drying, homogenization before encapsulation in spray-drying improves encapsulation efficiency during dehydration.Since the spray-drying encapsulation process does not require cryoprotectants, spray-drying can be used to produce dried NPs with high loading content.
This study reports the production of insulin-loaded NPs by cross-linking of chitosan and sodium tripolyphosphate using an ion gel method.Ion gelation is a preparation method that allows the production of nanoparticles through electrostatic interactions between two or more ionic species under certain conditions.Both freeze-drying and spray-drying techniques were used to dehydrate the optimized chitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles.After dehydration, their morphology was analyzed by SEM.Their recombination ability was evaluated by measuring their size distribution, surface charge, PDI, encapsulation efficiency, and loading content.The quality of resolubilized nanoparticles produced by different dehydration methods was also evaluated by comparing their insulin protection, release behavior, and cellular uptake efficacy.
The pH of the mixed solution and the ratio of chitosan and insulin are two key factors that affect the particle size and encapsulation efficiency (EE) of the final NPs, as they directly affect the ionotropic gelation process.The pH of the mixed solution was shown to be highly correlated with particle size and encapsulation efficiency (Fig. 1a).As shown in Fig. 1a, as pH increased from 4.0 to 6.0, the average particle size (nm) decreased and the EE increased significantly, while when the pH increased to 6.5, the average particle size started to increase and the EE remained unchanged.As the ratio of chitosan to insulin increases, the average particle size also increases.Furthermore, no change in EE was observed when nanoparticles were prepared at a mass ratio of chitosan/insulin higher than 2.5:1 (w/w) (Fig. 1b).Therefore, the optimal preparation conditions in this study (pH 6.0, chitosan/insulin mass ratio of 2.5:1) were used to prepare insulin-loaded nanoparticles for further study.Under this preparation condition, the average particle size of insulin nanoparticles was optimized to be 318 nm (Fig. 1c), the PDI was 0.18, the embedding efficiency was 99.4%, the zeta potential was 9.8 mv, and the insulin loading was 25.01% (m/m ).Based on transmission electron microscopy (TEM) results, the optimized nanoparticles were roughly spherical and discrete with relatively uniform size (Fig. 1d).
Parameter optimization of insulin nanoparticles: (a) the effect of pH on the mean diameter and encapsulation efficiency (EE) of insulin nanoparticles (prepared at 5:1 mass ratio of chitosan and insulin); (b) chitosan and Influence of mass ratio of insulin on the mean diameter and encapsulation efficiency (EE) of insulin NPs (prepared at pH 6); (c) particle size distribution of optimized insulin nanoparticles; (d) TEM micrograph of optimized insulin NPs .
It is well known that chitosan is a weak polyelectrolyte with a pKa of 6.5.It is positively charged in acidic media because its main amino group is protonated by hydrogen ions15.Therefore, it is often used as a carrier to encapsulate negatively charged macromolecules.In this study, chitosan was used to encapsulate insulin with an isoelectric point of 5.3.Since chitosan is used as a coating material, with the increase of its proportion, the thickness of the outer layer of the nanoparticles increases correspondingly, resulting in a larger average particle size.In addition, higher levels of chitosan can encapsulate more insulin.In our case, EE was highest when the ratio of chitosan and insulin reached 2.5:1, and there was no significant change in EE when the ratio kept increasing.
Besides the ratio of chitosan and insulin, pH also played a crucial role in the preparation of NPs.Gan et al. 17 studied the effect of pH on the particle size of chitosan nanoparticles.They found a continuous decrease in particle size until pH reached 6.0, and a significant increase in particle size was observed at pH > 6.0, which is consistent with our observations.This phenomenon is due to the fact that with increasing pH, the insulin molecule acquires a negative surface charge, thus, favoring electrostatic interactions with the chitosan/sodium tripolyphosphate (TPP) complex, resulting in small particle size and high EE .However, when the pH was adjusted to 6.5, the amino groups on chitosan were deprotonated, resulting in chitosan folding.Thus, high pH results in less exposure of amino ions to TPP and insulin, resulting in lower cross-linking, larger final average particle size and lower EE.
Analysis of the morphological properties of freeze-dried and spray-dried NPs can guide the selection of better dehydration and powder formation techniques.The preferred method should provide drug stability, uniform particle shape, high drug loading and good solubility in the original solution.In this study, to better compare the two techniques, insulin NPs with or without 1% mannitol were used during dehydration.Mannitol is used as a bulking agent or cryoprotectant in various dry powder formulations for freeze drying and spray drying.For the lyophilized insulin nanoparticles without mannitol, as shown in Figure 2a, a highly porous powder structure with large, irregular and rough surfaces was observed under scanning electron microscopy (SEM).Few discrete particles were detected in the powder after dehydration (Fig. 2e).These results indicated that most NPs were decomposed during freeze-drying without any cryoprotectant.For freeze-dried and spray-dried insulin nanoparticles containing 1% mannitol, spherical nanoparticles with smooth surfaces were observed (Fig. 2b,d,f,h).Insulin nanoparticles spray-dried without mannitol remained spherical but wrinkled on the surface (Fig. 2c).Spherical and wrinkled surfaces are further discussed in the release behavior and cellular uptake tests below.Based on the visible appearance of the dried NPs, both spray-dried NPs without mannitol and NPs freeze-dried and spray-dried with mannitol yielded fine NPs powders (Fig. 2f,g,h).The larger the surface area between the particle surfaces, the higher the solubility and therefore the higher the release rate.
Morphology of different dehydrated insulin NPs: (a) SEM image of lyophilized insulin NPs without mannitol; (b) SEM image of lyophilized insulin NPs with mannitol; (c) spray-dried insulin NPs without mannitol SEM image of ; (d) SEM image of insulin NPs spray-dried with mannitol; (e) image of lyophilized insulin NPs powder without mannitol; (f) image of lyophilized insulin NPs with mannitol; ( g) Image of spray-dried insulin NPs powder without mannitol; (h) image of spray-dried insulin NPs powder with mannitol.
During freeze-drying, mannitol acts as a cryoprotectant, keeping NPs in an amorphous form and preventing damage by ice crystals19.In contrast, there is no freezing step during spray drying.Therefore mannitol is not required in this method.In fact, spray-dried NPs without mannitol yielded finer NPs as previously described.However, mannitol can still act as a filler in the spray-drying process to give NPs a more spherical structure20 (Fig. 2d), which helps to obtain uniform release behavior of such encapsulated NPs.In addition, it is clear that some large particles can be detected in both freeze-dried and spray-dried insulin NPs containing mannitol (Fig. 2b,d), which may be due to the accumulation of mannitol in the particle core together with the encapsulated insulin. To.Chitosan layer.It is worth noting that in this study, in order to ensure that the spherical structure remains intact after dehydration, the ratio of mannitol and chitosan is kept at 5:1, so that a large amount of filler can also enlarge the particle size of the dried NPs..
Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy characterized the physical mixture of free insulin, chitosan, chitosan, TPP and insulin.All dehydrated NPs were characterized by using FTIR-ATR spectroscopy.Notably, band intensities of 1641, 1543 and 1412 cm-1 were observed in encapsulated NPs freeze-dried with mannitol and in NPs spray-dried with and without mannitol (Fig. 3).As previously reported, these increases in strength were associated with cross-linking between chitosan, TPP and insulin.Investigation of the interaction between chitosan and insulin showed that in the FTIR spectra of insulin-loaded chitosan nanoparticles, the chitosan band overlapped with that of insulin, increasing the carbonyl intensity (1641 cm-1) and amine (1543 cm-1) belt.The tripolyphosphate groups of TPP are linked to ammonium groups in chitosan, forming a band at 1412 cm-1.
FTIR-ATR spectra of free insulin, chitosan, physical mixtures of chitosan/TPP/insulin and NPs dehydrated by different methods.
Furthermore, these results are consistent with those shown in SEM, which showed that the encapsulated NPs remained intact both when sprayed and freeze-dried with mannitol, but in the absence of mannitol, only spray-drying produced encapsulated particles.In contrast, the FTIR-ATR spectral results of NPs freeze-dried without mannitol were very similar to the physical mixture of chitosan, TPP, and insulin.This result indicates that the cross-links between chitosan, TPP and insulin are no longer present in NPs freeze-dried without mannitol.The NPs structure was destroyed during freeze-drying without cryoprotectant, which can be seen in the SEM results (Fig. 2a).Based on the morphology and FTIR results of dehydrated insulin NPs, only lyophilized, spray-dried, and mannitol-free NPs were used for reconstitution experiments and mannitol-free NPs due to the decomposition of mannitol-free NPs during dehydration. discuss.
Dehydration is used for long-term storage and reprocessing into other formulations.The ability of dry NPs to reconstitute after storage is critical for their use in different formulations such as tablets and films.We noticed that the average particle size of the spray-dried insulin NPs in the absence of mannitol increased only slightly after reconstitution.On the other hand, the particle size of the spray-dried and freeze-dried insulin nanoparticles with mannitol increased significantly (Table 1).PDI and EE were not significantly changed (p > 0.05) after recombination of all NPs in this study (Table 1).This result indicates that most of the particles remained intact after redissolving.However, the addition of mannitol resulted in greatly reduced insulin loading of lyophilized and spray-dried mannitol nanoparticles (Table 1).In contrast, the insulin load content of NPs spray-dried without mannitol remained the same as before (Table 1).
It is well known that the loading of nanoparticles is critical when used for drug delivery purposes.For NPs with low loadings, very large amounts of material are required to reach the therapeutic threshold.However, the high viscosity of such high NP concentrations leads to inconvenience and difficulty in oral administration and injectable formulations, respectively 22 .In addition, insulin NPs can also be used to make tablets and viscous biofilms23, 24, which requires the use of large amounts of NPs at low loading levels, resulting in large tablets and thick biofilms that are not suitable for oral applications.Therefore, dehydrated NPs with high insulin load are highly desirable.Our results suggest that the high insulin load of mannitol-free spray-dried NPs can offer many attractive advantages for these alternative delivery methods.
All dehydrated NPs were kept in the refrigerator for three months.SEM results showed that the morphology of all dehydrated NPs did not change significantly during the three-month storage (Fig. 4).After reconstitution in water, all NPs showed a slight decrease in EE and released approximately a small amount (~5%) of insulin during the three-month storage period (Table 2).However, the average particle size of all nanoparticles increased.The particle size of NPs spray-dried without mannitol increased to 525 nm, while that of spray-dried and freeze-dried NPs with mannitol increased to 872 and 921 nm, respectively (Table 2).
Morphology of different dehydrated insulin NPs stored for three months: (a) SEM image of lyophilized insulin NPs with mannitol; (b) SEM image of spray-dried insulin nanoparticles without mannitol; (c) without mannitol SEM images of spray-dried insulin NPs.
Furthermore, precipitates were seen in the reconstituted insulin nanoparticles spray-dried with mannitol and freeze-dried (Fig. S2).This may be caused by large particles not properly suspending in the water.All the above results demonstrate that the spray drying technique can protect insulin nanoparticles from dehydration and that high loadings of insulin nanoparticles can be obtained without any fillers or cryoprotectants.
Insulin retention was tested in pH = 2.5 medium with pepsin, trypsin, and α-chymotrypsin to demonstrate the protective ability of NPs against enzymatic digestion after dehydration.The insulin retention of dehydrated NPs was compared with that of freshly prepared NPs, and free insulin was used as a negative control.In this study, free insulin showed rapid insulin elimination within 4 h in all three enzymatic treatments (Fig. 5a–c).In contrast, insulin elimination testing of NPs freeze-dried with mannitol and NPs spray-dried with or without mannitol showed significantly higher protection of these NPs against enzymatic digestion, which was similar to that of freshly prepared insulin NPs ( figure 1).5a-c).With the help of nanoparticles in pepsin, trypsin, and α-chymotrypsin, more than 50%, 60%, and 75% of insulin could be protected within 4 h, respectively (Fig. 5a–c).This insulin-protective ability may increase the chance of higher insulin absorption into the bloodstream25.These results suggest that spray drying with or without mannitol and freeze-drying with mannitol can preserve the insulin-protective ability of NPs after dehydration.
Protection and release behavior of dehydrated insulin NPs: (a) protection of insulin in pepsin solution; (b) protection of insulin in trypsin solution; (c) protection of insulin by α-chymotrypsin solution; ( d) The release behavior of dehydrated NPs in pH = 2.5 solution; (e) the release behavior of dehydrated NPs in pH = 6.6 solution; (f) the release behavior of dehydrated NPs in pH = 7.0 solution.
Freshly prepared and reconstituted dry insulin NPs were incubated in various buffers (pH = 2.5, 6.6, 7.0) at 37 °C, simulating the pH environment of the stomach, duodenum, and upper small intestine, to examine the effect of insulin on insulin resistance. Release behavior in different environments.Fragment of the gastrointestinal tract.At pH = 2.5, insulin-loaded NPs and resolubilized dry insulin NPs showed an initial burst release within the first one hour, followed by a slow release over the next 5 hours (Fig. 5d).This rapid release at the beginning is most likely the result of rapid surface desorption of protein molecules that are not fully immobilized in the internal structure of the particle.At pH = 6.5, insulin-loaded NPs and reconstituted dry insulin NPs showed a smooth and slow release over 6 h, as the pH of the test solution was similar to that of the NPs-prepared solution (Fig. 5e).At pH = 7, the NPs were unstable and almost completely decomposed within the first two hours (Fig. 5f).This is because deprotonation of chitosan occurs at higher pH, which results in a less compact polymer network and release of loaded insulin.
Furthermore, the insulin NPs spray-dried without mannitol showed a faster release profile than the other dehydrated NPs (Fig. 5d–f).As previously described, the reconstituted insulin NPs dried without mannitol showed the smallest particle size.Small particles provide a larger surface area, so most of the relevant drug will be at or near the particle surface, resulting in rapid drug release26.
The cytotoxicity of NPs was investigated by MTT assay.As shown in Figure S4, all dehydrated NPs were found to have no significant effect on cell viability at concentrations of 50–500 μg/ml, suggesting that all dehydrated NPs can be safely used to reach the therapeutic window.
The liver is the main organ through which insulin exerts its physiological functions.HepG2 cells are a human hepatoma cell line commonly used as an in vitro hepatocyte uptake model.Here, HepG2 cells were used to assess cellular uptake of NPs dehydrated using freeze-drying and spray-drying methods.Cellular uptake by confocal laser scanning using flow cytometry and vision following several hours of incubation with free FITC insulin at a concentration of 25 μg/mL, freshly prepared FITC insulin-loaded NPs and dehydrated FITC insulin-loaded NPs at equal insulin concentrations Quantitative microscopy (CLSM) observations were performed.Lyophilized NPs without mannitol were destroyed during dehydration and were not evaluated in this test.The intracellular fluorescence intensities of freshly prepared insulin-loaded NPs, lyophilized NPs with mannitol, and spray-dried NPs with and without mannitol (Fig. 6a) were 4.3, 2.6, 2.4, and 4.1-fold higher than the free ones.FITC-insulin group, respectively (Fig. 6b).These results suggest that encapsulated insulin is more potent in cellular uptake than free insulin, mainly due to the smaller size of the insulin-loaded nanoparticles produced in the study.
HepG2 cell uptake after 4 h incubation with freshly prepared NPs and dehydrated NPs: (a) Distribution of FITC-insulin uptake by HepG2 cells.(b) Geometric mean of fluorescence intensities analyzed by flow cytometry (n = 3), *P < 0.05 compared with free insulin.
Likewise, the CLSM images showed that the FITC fluorescence intensities of freshly prepared FITC-insulin-loaded NPs and FITC-insulin-loaded spray-dried NPs (without mannitol) were much stronger than those of the other samples (Fig. 6a).Furthermore, with the addition of mannitol, the higher viscosity of the solution increased the resistance to cellular uptake, resulting in decreased insulin proliferation.These results suggest that mannitol-free spray-dried NPs exhibited the highest cellular uptake efficiency because their particle size was smaller than that of freeze-dried NPs after re-dissolution.
Chitosan (average molecular weight 100 KDa, 75–85% deacetylated) was purchased from Sigma-Aldrich.(Oakville, Ontario, Canada).Sodium tripolyphosphate (TPP) was purchased from VWR (Radnor, Pennsylvania, USA).Recombinant human insulin used in this study was from Fisher Scientific (Waltham, MA, USA).Fluorescein isothiocyanate (FITC)-labeled human insulin and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich.(Oakville, Ontario, Canada).The HepG2 cell line was obtained from ATCC (Manassas, Virginia, USA).All other reagents were analytical or chromatographic grade.
Prepare a 1 mg/ml CS solution by dissolving it in double distilled water (DD water) containing 0.1% acetic acid.Prepare 1 mg/ml solutions of TPP and insulin by dissolving them in DD water and 0.1% acetic acid, respectively.The pre-emulsion was prepared with a polytron PCU-2-110 high speed homogenizer (Brinkmann Ind. Westbury, NY, USA).The preparation process is as follows: firstly, 2ml of TPP solution is added to 4ml of insulin solution, and the mixture is stirred for 30min and fully mixed.Then, the mixed solution was added dropwise to the CS solution through a syringe under high-speed stirring (10,000 rpm).The mixtures were kept under high-speed stirring (15,000 rpm) in an ice bath for 30 min, and they were adjusted to a certain pH to obtain cross-linked insulin NPs.To further homogenize and reduce the particle size of insulin NPs, they were sonicated for an additional 30 min in an ice bath using a probe-type sonicator (UP 200ST, Hielscher Ultrasonics, Teltow, Germany).
Insulin NPS were tested for Z-average diameter, polydispersity index (PDI) and zeta potential using dynamic light scattering (DLS) measurements using a Litesizer 500 (Anton Paar, Graz, Austria) by diluting them in DD water at 25°C.Morphology and size distribution were characterized by a Hitachi H7600 transmission electron microscope (TEM) (Hitachi, Tokyo, Japan), and images were subsequently analyzed using Hitachi imaging software (Hitachi, Tokyo, Japan).To assess the encapsulation efficiency (EE) and loading capacity (LC) of insulin NPs, the NPs were pipetted into ultrafiltration tubes with a molecular weight cut-off of 100 kDa and centrifuged at 500 x g for 30 min.Unencapsulated insulin in the filtrate was quantified using an Agilent 1100 Series HPLC system (Agilent, Santa Clara, California, USA) consisting of a quaternary pump, autosampler, column heater, and DAD detector.Insulin was analyzed by a C18 column (Zorbax, 3.5 μm, 4.6 mm × 150 mm, Agilent, USA) and detected at 214 nm.The mobile phase was acetonitrile and water, containing 0.1% TFA, gradient ratios from 10/90 to 100/0, and run for 10 minutes.The mobile phase was pumped at a flow rate of 1.0 ml/min.The column temperature was set to 20 °C.Calculate the percentages of EE and LC using the equations.(1) and Eq.(2).
Various CS/insulin ratios ranging from 2.0 to 4.0 were tested to optimize insulin NP.Different amounts of CS solution were added during the preparation, while the insulin/TPP mixture was kept constant.Insulin NPs were prepared in the pH range of 4.0 to 6.5 by carefully controlling the pH of the mixture after adding all solutions (insulin, TPP and CS).The EE and particle size of insulin nanoparticles were evaluated at different pH values ​​and CS/insulin mass ratios to optimize the formation of insulin NPs.
The optimized insulin NPs were placed on the aluminum container and covered with tissue tightened with some tape.Subsequently, the screwed containers were placed in a Labconco FreeZone freeze dryer (Labconco, Kansas City, MO, USA) equipped with a tray dryer.The temperature and vacuum pressure were set at -10 °C, 0.350 Torr for the first 2 h, and 0 °C and 0.120 Torr for the remaining 22 h of the 24 h to obtain dry insulin NPs.
Buchi Mini Spray Dryer B-290 (BÜCHI, Flawil, Switzerland) was used to generate encapsulated insulin.The selected drying parameters were: temperature 100 °C, feed flow 3 L/min, and gas flow 4 L/min.
Insulin NPs before and after dehydration were characterized using FTIR-ATR spectroscopy.Dehydrated nanoparticles as well as free insulin and chitosan were analyzed using a Spectrum 100 FTIR spectrophotometer (PerkinElmer, Waltham, Massachusetts, USA) equipped with a universal ATR sampling accessory (PerkinElmer, Waltham, Massachusetts, USA).Signal averages were obtained from 16 scans at a resolution of 4 cm2 in the frequency range of 4000-600 cm2.
Morphology of dry insulin NPs was assessed by SEM images of freeze-dried and spray-dried insulin NPs captured by a Helios NanoLab 650 Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) (FEI, Hillsboro, Oregon, USA). The main parameter used was voltage 5 keV and current 30 mA.
All dehydrated insulin NPs were redissolved in dd water.Particle size, PDI, EE and LC were tested again using the same method mentioned earlier to assess their quality after dehydration.The stability of anhydroinsulin NPs was also measured by testing the properties of the NPs after prolonged storage.In this study, all NPs after dehydration were stored in the refrigerator for three months.After three months of storage, NPs were tested for morphological particle size, PDI, EE and LC.
Dissolve 5 mL of reconstituted NPs in 45 mL containing simulated gastric fluid (pH 1.2, containing 1% pepsin), intestinal fluid (pH 6.8, containing 1% trypsin) or chymotrypsin solution (100 g/mL, in phosphate buffer, pH 7.8) to evaluate the efficacy of insulin in protecting NPs after dehydration.They were incubated at 37°C with agitation speed of 100 rpm.500 μL of the solution was collected at different time points and the insulin concentration was determined by HPLC.
The in vitro release behavior of freshly prepared and dehydrated insulin NPs was tested by the dialysis bag method (molecular weight cut-off 100 kDa, Spectra Por Inc.).Freshly prepared and reconstituted dry NPs were dialyzed in fluids at pH 2.5, pH 6.6, and pH 7.0 (0.1 M phosphate-buffered saline, PBS) to simulate the pH environment of the stomach, duodenum, and upper small intestine, respectively.All samples were incubated at 37 °C with continuous shaking at 200 rpm.Aspirate the fluid outside the 5 mL dialysis bag at the following times: 0.5, 1, 2, 3, 4, and 6 h, and immediately replenish the volume with fresh dialysate.Insulin contamination in the fluid was analyzed by HPLC, and the rate of insulin release from the nanoparticles was calculated from the ratio of free insulin released to total insulin encapsulated in the nanoparticles (Equation 3).
Human hepatocellular carcinoma cell line HepG2 cells were grown in 60 mm diameter dishes using Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin29.Cultures were maintained at 37°C, 95% relative humidity, and 5% CO2.For uptake assays, HepG2 cells were seeded at 1 × 105 cells/ml onto an 8-well Nunc Lab-Tek chamber slide system (Thermo Fisher, NY, USA).For cytotoxicity assays, they were seeded into 96-well plates (Corning, NY, USA) at a density of 5 × 104 cells/ml.
The MTT assay was used to evaluate the cytotoxicity of freshly prepared and dehydrated insulin NPs30.HepG2 cells were seeded in 96-well plates at a density of 5 × 104 cells/mL and cultured for 7 days prior to testing.Insulin NPs were diluted to various concentrations (50 to 500 μg/mL) in culture medium and then administered to cells.After 24 hours of incubation, cells were washed 3 times with PBS and incubated with medium containing 0.5 mg/ml MTT for an additional 4 hours.Cytotoxicity was assessed by measuring the enzymatic reduction of yellow tetrazolium MTT to purple formazan at 570 nm using a Tecan infinite M200 pro spectrophotometer plate reader (Tecan, Männedorf, Switzerland).
The cellular uptake efficiency of NPs was tested by confocal laser scanning microscopy and flow cytometry analysis.Each well of the Nunc Lab-Tek chamber slide system was treated with free FITC-insulin, FITC-insulin-loaded NPs, and reconstituted 25 μg/mL of dehydrated FITC-insulin NPs at the same concentration and incubated for 4 hours.Cells were washed 3 times with PBS and fixed with 4% paraformaldehyde.Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).Insulin localization was observed using an Olympus FV1000 laser scanning/two-photon confocal microscope (Olympus, Shinjuku City, Tokyo, Japan).For flow cytometry analysis, the same concentrations of 10 μg/mL free FITC-insulin, FITC-insulin-loaded NPs, and resolubilized dehydrated FITC-insulin NPs were added to 96-well plates seeded with HepG2 cells and incubated for 4 hours .After 4 h of incubation, cells were removed and washed 3 times with FBS.5 × 104 cells per sample were analyzed by a BD LSR II flow cytometer (BD, Franklin Lakes, New Jersey, United States).
All values ​​are expressed as mean ± standard deviation.Comparisons between all groups were assessed using one-way ANOVA or t-test by IBM SPSS Statistics 26 for Mac (IBM, Endicott, New York, USA) and p < 0.05 was considered statistically significant.
This study demonstrates the flexibility and ability of spray drying to dehydrate cross-linked chitosan/TPP/insulin nanoparticles with better reconstitution compared to standard freeze-drying methods using bulking agents or cryoprotectants capacity and higher load capacity.The optimized insulin nanoparticles yielded an average particle size of 318 nm and an encapsulation efficiency of 99.4%.SEM and FTIR results after dehydration showed that the spherical structure was maintained only in spray-dried NPs with and without mannitol and lyophilized with mannitol, but lyophilized NPs without mannitol were decomposed during dehydration.In the reconstitution ability test, insulin nanoparticles spray-dried without mannitol showed the smallest mean particle size and the highest loading upon reconstitution.The release behaviors of all these dehydrated NPs showed that they were rapidly released in solutions of pH = 2.5 and pH = 7, and very stable in solution of pH = 6.5.Compared to other redissolved dehydrated NPs, the NPs spray-dried without mannitol showed the fastest release.This result is consistent with that observed in the cellular uptake assay, as spray-dried NPs in the absence of mannitol almost completely maintained the cellular uptake efficiency of freshly prepared NPs.These results suggest that dry insulin nanoparticles prepared by mannitol-free spray drying are most suitable for further processing into other anhydrous dosage forms, such as oral tablets or bioadhesive films.
Due to intellectual property issues, the datasets generated and/or analyzed during the current study are not publicly available, but are available from the respective authors upon reasonable request.
Kagan, A. Type 2 diabetes: social and scientific origins, medical complications, and implications for patients and others.(McFarlane, 2009).
Singh, AP, Guo, Y., Singh, A., Xie, W. & Jiang, P. The development of insulin encapsulation: is oral administration now possible?J. Pharmacy.bio-pharmacy.reservoir.1, 74–92 (2019).
Wong, CY, Al-Salami, H. & Dass, CR Recent advances in oral insulin-loaded liposome delivery systems for the treatment of diabetes.Interpretation.J. Pharmacy.549, 201–217 (2018).