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Synthesis and characterization of dendro-PLGA nanoconjugate for protein stabilization
Accepted Manuscript Title: Synthesis and characterization of dendro-PLGA nanoconjugate for protein stabilization “Your article is registered as a regular item and is being processed for inclusion in a regular issue of the journal. If this is NOT correct and your article belongs to a Special Issue/Collection please contact [email protected] immediately prior to returning your corrections.”–> Author: Amit Tiwari Prashant Kesharwani Virendra Gajbhiye Narendra K. Jain PII: DOI: Reference:
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Colloids and Surfaces B: Biointerfaces
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Synthesis and characterization of dendro-PLGA nanoconjugate for protein stabilization Amit Tiwari1, Prashant Kesharwani1,2*, Virendra Gajbhiye1, Narendra K. Jain1* 1
Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour Central University, Sagar (MP) 470003, INDIA 2 Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy & Health Sciences, Wayne State University, Detroit (MI) 48201, USA
*Address for correspondence Dr. Prashant Kesharwani C/O Prof. N. K. Jain Pharmaceutics Research Laboratory Department of Pharmaceutical Sciences Dr. Hari Singh Gour Central University SAGAR 470 003 [INDIA] Tel/Fax: +91-7582-265055 Email: [email protected] (N. K. Jain); [email protected]; [email protected] (Prashant Kesharwani) Disclosures: There is no conflict of interest and disclosures associated with the Highlights ► manuscript.
Highlights
The present study was aimed to development of Dendro-PLGA nano-conjugate for protection of insulin from degradation as well as sustained release of it from nanocarriers. The novel dendro PLGA nanoconjugate not only stabilize the insulin but also work as a sustained release reservoir for insulin which reduces the frequency of dosage and side effect associate with denatured protein.
ABSTRACT The present investigation was aimed to develop the Dendro-PLGA nanoconjugate (DPNC) for protection of insulin from degradation as well as its sustained release from nano-formulation. DPNC formulation was synthesized by 1-Ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC) coupling reaction and therapeutic efficacy of encapsulated protein (insulin) was measured by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Fourier Transform Infrared spectroscopy (FTIR) and Circular dichorism (CD) spectroscopy. We have also demonstrated the ability of DPNC formulation to prevent the native conformation of insulin within the system by comparing the amount of free protein with the protein extracted from this system. Stability study further revealed that as compared to free protein, DPNC formulation was more efficient to stabilize the protein. Additionally, in vivo data of protein loaded system in rats showed that DPNC formulation can able to maintain the native structure of insulin and hence retain therapeutic efficacy of protein. The novel dendro-PLGA nanoconjugate not only stabilize the insulin but also work as sustained release reservoir for insulin which reduces the frequency of dosage and side effect associate with denatured protein. Keywords Insulin, PPI Dendrimer, PLGA, Protein, Stability.
1. Introduction Proteins are complex three-dimensional molecules, whose functionality depends on their structure [1]. They are prone to chemical (e.g., deamidation, oxidation) and physical (aggregation, precipitation, and adsorption) changes during storage [1-4]. The mechanisms by which proteins undergo structural alterations are protein-specific; however, temperature and moisture are main factors that decrease the stability of proteins [2,3,5,6]. A carrier capable of encapsulating proteins, minimizing the mechanisms of degradation and maximizing the in vivo activity, and providing controlled release that can be maintained the therapeutic activity of proteins over a prolonged period of time.
The polymer that has received the most attention as a protein delivery vehicle is poly (d,l-lactide-co-glycolic acid) (PLGA). PLGA has been used to encapsulate and maintaining the therapeutic activity of numerous model and recombinant proteins, such as bovine serum albumin (BSA) [7-14], lysozyme [13,15,16], recombinant human growth hormone (rhGH) [17-20], and recombinant human insulin like growth factor-1 (rhIGF-I) [21-23]. Major problem with PLGA is that, as it degrades, the pH within the polymeric device drops significantly [24], which can be detrimental to the protein. This problem can be overcome by the co encapsulation of basic compounds, which have proven to help stabilize the encapsulated protein [7]. Due to their sophisticated and delicate structure, protein drugs are highly susceptible of losing activity during polymer based sustained-release formulation process and in long-term storage [25-30]. On the other hand, the preparation of protein loaded microspheres is associated with some problems. Only a limited fraction of the protein may be entrapped during the incorporation process. Recently it was observed that the bioactivity of some proteins was severely reduced after incorporation into microsphere [31-33]. This effect is probably the result of the chemical and mechanical stress imposed during microsphere preparation causing structural changes in the protein. Dendrimers are highly branched and reactive three-dimensional polymers, with all bonds emanating from a central core. They have many attractive features like nanoscopic size, highly controllable molecular weight, large number of readily accessible terminal functional groups and possibility of encapsulating a guest molecule in internal cavities give dendrimers a distinct edge over other polymers for the delivery of drugs [34]. Based on these merits of dendrimer, we thus engineered Dendro-PLGA nanoconjugate (DPNC) for protection of insulin from degradation as well as its sustained release from nano-formulation.
2. Materials and Methods 2.1. Materials Ethylene diamine (EDA) and acrylonitrile (ACN) were purchased from CDH, India. Raney Nickel was purchased from Merck, India. Human insulin was obtained as
generous
gift
from
Biocon
Ltd.
Banglore,
India.
1-Ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC) and cellulose dialysis bag (MWCO 12-14 kDa), were purchased from Himedia, India. PLGA (MW 8000) was obtained as generous gift from Sun Pharma Advanced Research Company Ltd. Vadodara, India. All the other chemicals used were of analytical grade. 2.2. Synthesis and characterization of 5.0G poly(propyleneimine) (PPI) dendrimers 5.0G PPI dendrimers were synthesized by earlier reported divergent method [35,36]. Briefly, EDA was used as the initiator core. ACN was added to it in a double Michael addition reaction to produce half-generation (–CN terminated) dendrimer. Excess ACN was removed as water azeotrope by vacuum distillation (rotary flask evaporator, Superfit, India). CN-terminated dendrimer was heterogeneously hydrogenated using Raney nickel catalyst to produce full-generation (–NH2 terminated) dendrimer. The reaction sequence was repeated cyclically to produce PPI dendrimer up to fifth generation. Characterization of PPI dendrimer was carried out by using 1H NMR spectroscopy, IR spectroscopy and TEM microscopy. 2.3. Conjugation of PLGA to PPI dendrimer and its characterization The conjugation of PLGA was performed following the activation of end functional groups of PLGA. The activation of PLGA was carried out by converting it to dicarboxylic acid derivatives and subsequently to N-hydroxysuccinimide (NHS) ester [37] with slight modification. The polymer PLGA (62.5 µM) (MW-8000) was dissolved in 20 ml of dichloromethane (DCM) and 52 µM EDC was added in this solution. The resulting mixture was stirred overnight to obtain a clear yellow solution.
5.7µM of 5.0G PPI dendrimers was dissolved in 20.0 ml of methanol and in this solution activated PLGA solution was added. The resultant sample was stirred continuously 48 hours by using a magnetic stirrer in dark at room temperature. The final product was dialyzed by dialysis membrane (MWCO 12-14 kDa, Himedia, India) for 24 hours to remove free PLGA and unconjugated dendrimer. Excess solvent in this dialyzed product was removed by rotary vacuum evaporator (Scheme 1). Characterization of Dendro PLGA nanoconjugate was carried out by using FTIR, nuclear magnetic resonance (1H NMR) spectroscopy, and transmission electron microscopic (TEM) microscopy. 2.4. Drug loading and entrapment efficiency The known molar concentrations of PPI 5.0G dendrimer and DPNC were dissolved separately in phosphate buffer saline (PBS) (pH-7.4) and mixed with insulin solution in hydrochloride acid solution (0.1 M) which was also dissolved in same solvent. The mixed solutions were incubated with slow magnetic stirring (50 rpm) using teflon beads for 24 h at 2-80C. This preparation was taken and dialyzed in cellulose dialysis membrane (MWCO 12-14 kDa, Himedia, India) against PBS (pH7.4) under sink condition for 15 min to remove the unentrapped drug from the formulation, which was then estimated spectrophotometrically (λmax 562 nm) (UV 1601, Shimadzu, Japan)
by using bicinchoninic acid assay (BCA) kit to determine
indirectly the amount of loaded drug [38]. 2.5. In vitro drug release The in vitro drug release studies were carried out to determine the release of insulin from plain PPI dendrimer and DPNC by equilibrium dialysis (MWCO 12-14 kDa, Himedia, India) method [39]. Both drug loaded plain PPI dendrimer and DPNC (2 ml) were placed in the dialysis sacs separately. The sacs were hermetically sealed, and suspended in 30 ml of release medium PBS (pH-7.4), on magnetic stirrer
maintained at 370C. At predetermined time intervals; 1 ml of the release medium was withdrawn from both release assembly and this sample was used for the estimation of free insulin content. Every time, the amount of aliquot was removed and replaced by the equal amount of fresh PBS (pH-7.4) to maintain the sink condition during the experiment. The drug release was determined after appropriate dilution, using BCA kit at 562 nm. 2.6. Comparison of hemolytic toxicity of 5.0G PPI dendrimer and PLGA conjugated 5.0 G PPI dendrimer The RBC suspension was obtained as per the well-known and reported procedure for hemolytic studies [40,41]. In brief, the RBC suspension (5% hematocrit) of the human blood was collected in HiAnticlot blood collection vials (Himedia Labs, India). 0.5 ml of suitably diluted PLGA conjugated and nonconjugated PPI 5.0G were added to 4.5 ml of normal saline and incubated for 1hr with RBC suspension. This allowed comparison of the hemolysis data of the dendrimer and PLGA conjugated dendritic architectures to assess the effect of PLGA conjugation on hemolysis. After centrifugation, supernatants were taken and diluted with an equal volume of normal saline and absorbance was measured at 540 nm. RBC suspension was added to 5 ml of 0.9% NaCl solution (normal saline) and 5 ml distilled water, respectively to obtain 0 and 100% hemolysis. The degree of hemolysis was determined by the following equation:
ABs % Haemolysis = × 100 AB 100 where, ABs = Absorbance for the sample; and AB100 = Absorbance for control without formulation 2.7. Stability studies of 5.0 G PPI dendrimer and dendro PLGA nanoconjugate
Insulin loaded PPI dendrimer and dendro PLGA nanoconjugate as exposed to accelerated conditions of temperature and light. The formulation was taken in different vials and stored in dark (amber color vials) and in light (colorless vials) at 0ºC, room temperature and 50±2ºC in thermostatically controlled oven for a period of 5 weeks. The samples were analyzed every week for any precipitation, turbidity, crystallization, color change, consistency and drug leakage. The data obtained was used for the analysis of any physical and chemical degradation, the required storage conditions and the precautions required for storage. The samples were initially clear and transparent at 0ºC. The loss of drug from the formulation was ascertained after storage at accelerated conditions. The known amount of formulation was kept in benzoylated cellulose tubing (Sigma, USA) and dialyzed across the tubing. The external medium [PBS (pH-7.4)] was monitored for the content of the drugs spectrophotometrically. The percentage increase in drug release from the formulation was used to analyze the effects of accelerated conditions of storage on the formulations. Integrity of encapsulated drug was measured by SDS-PAGE, FTIR and CD spectroscopy. 2.8. In vivo studies The male albino rats (Sprague Dawley strain, 120±5 g) were used for in vivo experimental study. All the animal studies were conducted in accordance with the protocol approved by the Institutional Animal Ethical Committee of Dr. H. S. Gour University, Sagar (M.P.), India (Reg. No. 379/01/ab/CPCSEA). The animals were divided into 4 groups: Group 1 Control, Group 2 Plane insulin, Group 3 insulin loaded PPI dendrimer, Group 4 insulin loaded DPNC. Diabetes was induced in albino rats by single intraperitoneal injection of streptozocin (50 mg/kg). Both insulin loaded preparation DPNC, PPI dendrimer dissolved in PBS (pH-7.4) and plain insulin was also dissolved with PBS (pH-7.4) in the dose of 4 IU/kg body weight of rat and
administered through intravenous (IV) route in the albino rats of respective groups excluding control group. The control group was injected with PBS (pH-7.4). Blood glucose level of all animals was checked just before starting experiment by taking blood (0.1 mL) from the retro orbital plexus of the rats. Following the treatment, blood sample were collected from the retro orbital plexus of rats at predefined time intervals 0, 0.25, 0.5, 1, 2, 3, 4, 8, 12, 16 and 24 hr, and analyzed for glucose level in blood. 3. Results 3.1. Synthesis of PPI dendrimer Synthesis of 0.5G PPI was confirmed by IR peaks, mainly of nitrile at 2244.3 cm-1 and CH2 bending at 1467.7 cm-1 (Fig. 1a). All the nitrile terminal 0.5G PPI got converted into (NH2)4, which was confirmed by IR of PPI 1.0G that exhibited major peak at 3427.3 cm-1 of amine (N-H stretch) (Fig. 1b). Likewise, IR peaks also confirmed synthesis of 5.0G PPI dendrimers. The main peaks are of C-C bend (1106.6 cm-1); C-N stretch (1312.4 cm-1); C-H bend (1410.4 cm-1, 1463.2 cm-1); N-H deflection of amine (1665.5 cm-1) and primary amine at 3409.8 cm-1 (N-H stretch), confirming nitrile terminal groups of dendrimer were converted to amine terminals (Fig. 1c). The synthesis was also confirmed by proton NMR by the obtained major peaks and shifts. NMR of 5.0G PPI dendrimer revealed peaks of alkane were obtained between 0.6-1.25 ppm while peaks of alkyl amine were obtained between 1.3-1.9 ppm. Incompletely cyanoethyleted dendrimer was also evident between 3.2 to 3.8 ppm with integral value 37.86 (NCH2CH2-CN). Substituted alkyl amines exhibited peak between 3.2-3.8 ppm and primary amines exhibited peak at 7.99 ppm (Fig. 1d). TEM images of 5.0G PPI dendrimer proved their nanometric size (Fig. 1e). 3.2. Conjugation of PLGA to PPI dendrimer
The conjugation of PLGA to PPI dendrimer was confirmed by IR, NMR and TEM microscopy. The FTIR analysis of the conjugated system shows the characteristic peak of secondary amide at 3358.1 cm-1, with the additional peak of CH stretch at 2956.2 cm-1 and C=O stretch at 1667.0 cm-1 were observed (Fig. 2a). NMR spectroscopy results also prove the conjugation of PLGA with PPI dendrimer. Peak of ether linkage appears at 3.48-3.65 ppm, which proves the conjugation. The characteristic peak of amide linkage appeared near 7.27 ppm, which was absent in 5.0 G PPI dendrimer (Fig. 2b). The electron microscopic analysis of conjugated system displayed nanometric sized units, as evident from TEM. The increase in size of conjugated system also provides a strong evidence of the formation of DPNC (Fig. 2c). 3.3. Hemolytic toxicity The toxicity is due to the polycationic nature of the PPI dendrimers. However PLGA conjugation of dendrimers was found to decrease the hemolysis of the RBC considerably at all concentrations due to shielding or coating of the charged quaternary ammonium ion that is generally formed on the amine terminated whole generations of PPI dendrimers, responsible for hemolysis. The whole generation of amine-terminated charged 5.0G PPI dendrimers showed hemolytic toxicity 1.585±0.061, 2.181±0.063 and 3.443±0.036 at 0.01, 0.03 and 0.05 % w/v concentrations, respectively. However, the conjugation of PLGA to the dendrimers was found to have decreased the hemolysis of the RBCs significantly to 0.621±0.052, 1.145±0.0.065 and 1.660±0.083 at 0.01, 0.03 and 0.05 % w/v concentrations, respectively (Fig. 3). This was due to the inhibition of interaction of RBCs with the charged quaternary ammonium ion as determined by interaction with RBCs. These data can also favor the enhanced in vivo safety by our developed PLGA conjugated dendrimer.
3.4. Drug loading and entrapment efficiency The drug loading was performed by dialysis method using cellulose membrane (MWCO 12-14 kDa, Himedia, India) and un-entrapped drug was determined spectrophotometrically .The dendritic formulation was retained in the membrane due to its molecular weight and hyper branched polymeric spherical constitution while the free drug easily comes out of the membrane. The drug entrapment and retention is low in case of non conjugated dendrimer as compared to the conjugate system. This may be due to the covering of peripheral portion of dendrimer by PLGA polymer, which was responsible for the steric hindrance and as a consequence drug loading was improved. The entrapment efficiency of the system increased from PPI (16%) to DPNC (70%). 3.5. In vitro drug release The results of in vitro release of insulin from PPI dendrimer and DPNC are presented in Fig. 3a. The in vitro drug release profile shows that there is a slower release rate of insulin from DPNC as compared to PPI dendrimer. While the PPI completely releases the drug by 12 h, the PPI-based nanoconjugate, prolonged the release rate up to 36 h. This fact can once again be explained on the basis of steric hindrance at the periphery, which slows down and hence prolongs the release of insulin from DPNC as compared to PPI dendrimer (Fig. 4a). Once the therapeutic action has been achieved the conjugates may undergo renal clearance in fraction [42]. 3.6. Stability studies of PLGA conjugated dendrimer formulations The stability study carried out on the drug-PLGA conjugated dendrimer complexes at various accelerated conditions of temperature (0°C, RT, and 50°C) and light showed that the dendritic formulations are stable even at higher temperature if kept in dark (amber color vials) but are unstable in presence of light (colorless vials). There was change in color and precipitation noted after five weeks when kept at 50°C
in presence of light but no such change was observed at the same temperature in dark (Fig. 4b). However, there was drug loss from the formulation observed at higher temperature and it was found to be even greater in the presence of light. This may be due to higher reaction kinetics in presence of light at high temperature. The dendritic structures are supposed to be more open at higher temperature and this change in surface characteristics might cause the conformational changes in the structure and release of drug. SDS-PAGE of insulin showed that the drug retained native conformation after processing since the peak of insulin recovered from DPNC was matched with authentic sample of insulin (Fig. 5). Secondary structure of insulin after processing has been confirmed by FTIR spectroscopy (Fig. 6a). CD spectroscopy has been also carried out to further to confirm the preservation of secondary structure of insulin which is necessary for the protein therapeutic activity. CD spectrum of plain insulin showed 2 minima at 208 and 222 nm. These data was in close agreement with results presented by previous studies [43]. There was minor deviation in the negative maximum at about 220 nm for insulin recovered from DPNC. The secondary structure for insulin (native conformation) may have slightly changed after processing. The possible explanation for such observation would be the shear forces which acted during the formulation process, though not representing denaturation or loss of insulin activity (Fig. 6b). 3.7. In vivo studies Insulin loaded DPNC exhibited the longest time to decrease the blood glucose levels; while the insulin loaded PPI dendrimer exhibited the intermediate time, i.e. between insulin loaded DPNC and insulin suspension formulation, to decrease blood glucose level. Finally these results show that the insulin loaded DPNC exhibit the slower onset of glucose lowering over prolonged time period. This result also indicate
that DPNC stabilize the insulin during processing which is a major drawback associated with the market insulin formulation (Fig. 7). 4. Discussion Protein and peptides makes very important group of therapeutic agents but their instability during processing and storage damage their native structure which reduces their biological activity, inducing aggregation and render the proteins immunogenic, leading ultimately to precipitation [44]. The use of nanocarriers not only protects the proteins from different adverse conditions during storage but also act as a reservoir for control release of proteins. In present study, we synthesized a novel nanoconjugate system for the stabilization as well as delivery of protein. Dendrimers are a class of highly branched spherical polymeric systems. Two types of dendrimers, the polyamidoamine (PAMAM) [45] and PPI are commercially used [46]. Both types are highly soluble in aqueous solutions and have a unique surface covered by primary amino groups [47]. Due to their well-defined monodispersity, controlled architecture, nanosize (20-40 nm), and shape, they are often considered most promising vehicles for the stabilization and delivery of drugs, proteins, and other therapeutic agents [48-51]. Therefore, in a short time, they have emerged as the most promising drug delivery carriers. In this work, we conjugate the PLGA polymer to 5.0 G PPI dendrimer to form DPNC for the stabilization of protein. The drug entrapment and retention were low in case of non conjugated dendrimer as compared to the conjugate system. This might be due to the covering of peripheral portion of dendrimer by PLGA polymer, which was responsible for the steric hindrance and as a consequence drug loading was improved. The in vitro release of insulin from DPNC showed controlled release of drug as compared to insulin loaded unconjugated dendrimer in PBS (pH-7.4). This may be due to the
entrapment of insulin molecule in between long PLGA chain, possibly due to the electrostatic, hydrophobic interaction or hydrogen bonding. The hemolytic toxicity of the unconjugated PPI dendrimer as well as DPNC was carried out to determine the interaction between dendrimer and RBCs. The toxicity of the unconjugated PPI dendrimer is due to the polycationic nature of the PPI dendrimer. The modification of cationic dendrimers with PLGA shields the positive charge on the dendrimer surface as in case of PEGylation and leads to a decrease in cytotoxicity [52]. The stability study carried out on the insulin loaded PPI dendrimer and DPNC at various accelerated conditions of temperature (00C, RT, 500C) and light showed that the dendritic formulations were stable even at higher temperature if they are kept in dark (amber color vials), but unstable in the presence of light (colorless vials). The drug loss from the dendritic formulation at higher temperature and in presence of light was found to be more, as compared to room temperature and in dark, due to the destabilization and opening of dendritic structure, causing the loss of drug to a greater extent. Integrity of insulin recovered from DPNC confirmed by SDS-PAGE, FTIR and CD spectroscopy. Results obtained from these methods showed that the secondary structure for insulin (native conformation) retained after processing which are necessary for protein activity. In vivo results suggest that the insulin loaded DPNC exhibits slower onset of action and reduction in blood glucose level was found to be prolonged time period. These results also indicate that DPNC stabilizes the insulin during processing which is a major drawback associated with the market formulations of insulin. 5. Conclusions
Dendrimers are used as carrier macromolecules for many bioactives agents due to its targeting, imaging, and drug delivery property. When terminal amino group of PPI dendrimer are shielded by a polymer, then the toxicity of dendrimer is reduced and drug loading efficiency is also increased. The Dendro PLGA nanoconjugate retained the native structure of protein during processing which was confirmed by the SDS PAGE analysis, FTIR spectroscopy and CD spectroscopy. Non conjugated dendrimer showed more leakage of protein in adverse conditions as compared to the PLGA conjugated dendrimer. Furthermore the in vivo results of protein loaded DPNC also proved the retention of therapeutic activity of protein. Declaration of interest The authors report no conflicts of interest in this work. Acknowledgement Mr. Amit Kumar Tiwari acknowledges the All India Council of Technical Education, New Delhi, India for the grant of Junior Research Fellowship and Bicon Ltd., Banglore (India) for providing the gift sample of Human insulin. References [1] S.P. Schwendeman, M.A. Klibanov, R. Langer, M.R. Brandon. in: S. Cohen, H. Bernstein (Eds.), Microparticulate systems for the delivery of proteins and vaccines, Marcel Dekker, New York (1996) 1-49. [2] S.L. Nail. in: K. Park (Ed.), Controlled drug delivery challenges and strategies, American Chemical Society, Washington, DC (1997) 185-203. [3] Schoneich C, Hageman MJ, Borchardt RT. in: K. Park (Ed.), Controlled drug delivery challenges and strategies, American Chemical Society, Washington, DC (1997) 205-27. [4] J.L. Cleland, M.F. Powell, S.J. Shire. The development of stable protein formulations: a close look at protein aggregation, deamidation, and oxidation. Crit Rev Ther Drug Carr Syst 10 (1993) 307-77.
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Scheme 1. Schematic representation for conjugation of PPI 5.0G dendrimer to PLGA. Fig. 1. FTIR spectrum of (a) 0.5G (b) 1.0G (c) 5.0G PPI dendrimer; (d) NMR spectrum of 5.0G PPI dendrimer; (e) TEM photograph of 5.0G PPI dendrimer. Fig. 2. (a) FTIR (b) NMR spectrum (c) TEM photograph of PLGA conjugated dendrimer.
Fig. 3. Hemolytic toxicity profile of plain dendrimer and different PLGA-dendritic conjugates (n = 3). Fig. 4. (a) Cumulative percent drug release from dendritic formulations in PBS 7.4 as recipient medium (n = 3); (b) Percent drug leakage from drug loaded DPNC at different storage condition up to 5 weeks (n = 6) Fig. 5. SDS –PAGE after processing [Lane 1-molecular marker in kDa (180, 116, 58, 48.5, 36.5, 26.6, 10); Lane 2-Insulin; Lane 3-Insulin recovered from PPI dendrimer, Lane 4 – Insulin recovered from DPNC]. Fig. 6. (a) FTIR (b) CD-Spectra of insulin obtained from DPNC. Fig. 7. Blood glucose level in diabetic rats after administration of different formulations containing insulin equivalent to 4 IU/kg.
3_Graphical abstract .
Figure 1 .
Figure 2 .
Figure 3 .
Figure 4 .
Figure 5 .
Figure 6 .
Figure 7 .
S0927-7765(15)30029-1 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.06.064 COLSUB 7194
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Synthesis and characterization of dendro-PLGA nanoconjugate for protein stabilization Amit Tiwari1, Prashant Kesharwani1,2*, Virendra Gajbhiye1, Narendra K. Jain1* 1
Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour Central University, Sagar (MP) 470003, INDIA 2 Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy & Health Sciences, Wayne State University, Detroit (MI) 48201, USA
*Address for correspondence Dr. Prashant Kesharwani C/O Prof. N. K. Jain Pharmaceutics Research Laboratory Department of Pharmaceutical Sciences Dr. Hari Singh Gour Central University SAGAR 470 003 [INDIA] Tel/Fax: +91-7582-265055 Email: [email protected] (N. K. Jain); [email protected]; [email protected] (Prashant Kesharwani) Disclosures: There is no conflict of interest and disclosures associated with the Highlights ► manuscript.
Highlights
The present study was aimed to development of Dendro-PLGA nano-conjugate for protection of insulin from degradation as well as sustained release of it from nanocarriers. The novel dendro PLGA nanoconjugate not only stabilize the insulin but also work as a sustained release reservoir for insulin which reduces the frequency of dosage and side effect associate with denatured protein.
ABSTRACT The present investigation was aimed to develop the Dendro-PLGA nanoconjugate (DPNC) for protection of insulin from degradation as well as its sustained release from nano-formulation. DPNC formulation was synthesized by 1-Ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC) coupling reaction and therapeutic efficacy of encapsulated protein (insulin) was measured by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Fourier Transform Infrared spectroscopy (FTIR) and Circular dichorism (CD) spectroscopy. We have also demonstrated the ability of DPNC formulation to prevent the native conformation of insulin within the system by comparing the amount of free protein with the protein extracted from this system. Stability study further revealed that as compared to free protein, DPNC formulation was more efficient to stabilize the protein. Additionally, in vivo data of protein loaded system in rats showed that DPNC formulation can able to maintain the native structure of insulin and hence retain therapeutic efficacy of protein. The novel dendro-PLGA nanoconjugate not only stabilize the insulin but also work as sustained release reservoir for insulin which reduces the frequency of dosage and side effect associate with denatured protein. Keywords Insulin, PPI Dendrimer, PLGA, Protein, Stability.
1. Introduction Proteins are complex three-dimensional molecules, whose functionality depends on their structure [1]. They are prone to chemical (e.g., deamidation, oxidation) and physical (aggregation, precipitation, and adsorption) changes during storage [1-4]. The mechanisms by which proteins undergo structural alterations are protein-specific; however, temperature and moisture are main factors that decrease the stability of proteins [2,3,5,6]. A carrier capable of encapsulating proteins, minimizing the mechanisms of degradation and maximizing the in vivo activity, and providing controlled release that can be maintained the therapeutic activity of proteins over a prolonged period of time.
The polymer that has received the most attention as a protein delivery vehicle is poly (d,l-lactide-co-glycolic acid) (PLGA). PLGA has been used to encapsulate and maintaining the therapeutic activity of numerous model and recombinant proteins, such as bovine serum albumin (BSA) [7-14], lysozyme [13,15,16], recombinant human growth hormone (rhGH) [17-20], and recombinant human insulin like growth factor-1 (rhIGF-I) [21-23]. Major problem with PLGA is that, as it degrades, the pH within the polymeric device drops significantly [24], which can be detrimental to the protein. This problem can be overcome by the co encapsulation of basic compounds, which have proven to help stabilize the encapsulated protein [7]. Due to their sophisticated and delicate structure, protein drugs are highly susceptible of losing activity during polymer based sustained-release formulation process and in long-term storage [25-30]. On the other hand, the preparation of protein loaded microspheres is associated with some problems. Only a limited fraction of the protein may be entrapped during the incorporation process. Recently it was observed that the bioactivity of some proteins was severely reduced after incorporation into microsphere [31-33]. This effect is probably the result of the chemical and mechanical stress imposed during microsphere preparation causing structural changes in the protein. Dendrimers are highly branched and reactive three-dimensional polymers, with all bonds emanating from a central core. They have many attractive features like nanoscopic size, highly controllable molecular weight, large number of readily accessible terminal functional groups and possibility of encapsulating a guest molecule in internal cavities give dendrimers a distinct edge over other polymers for the delivery of drugs [34]. Based on these merits of dendrimer, we thus engineered Dendro-PLGA nanoconjugate (DPNC) for protection of insulin from degradation as well as its sustained release from nano-formulation.
2. Materials and Methods 2.1. Materials Ethylene diamine (EDA) and acrylonitrile (ACN) were purchased from CDH, India. Raney Nickel was purchased from Merck, India. Human insulin was obtained as
generous
gift
from
Biocon
Ltd.
Banglore,
India.
1-Ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC) and cellulose dialysis bag (MWCO 12-14 kDa), were purchased from Himedia, India. PLGA (MW 8000) was obtained as generous gift from Sun Pharma Advanced Research Company Ltd. Vadodara, India. All the other chemicals used were of analytical grade. 2.2. Synthesis and characterization of 5.0G poly(propyleneimine) (PPI) dendrimers 5.0G PPI dendrimers were synthesized by earlier reported divergent method [35,36]. Briefly, EDA was used as the initiator core. ACN was added to it in a double Michael addition reaction to produce half-generation (–CN terminated) dendrimer. Excess ACN was removed as water azeotrope by vacuum distillation (rotary flask evaporator, Superfit, India). CN-terminated dendrimer was heterogeneously hydrogenated using Raney nickel catalyst to produce full-generation (–NH2 terminated) dendrimer. The reaction sequence was repeated cyclically to produce PPI dendrimer up to fifth generation. Characterization of PPI dendrimer was carried out by using 1H NMR spectroscopy, IR spectroscopy and TEM microscopy. 2.3. Conjugation of PLGA to PPI dendrimer and its characterization The conjugation of PLGA was performed following the activation of end functional groups of PLGA. The activation of PLGA was carried out by converting it to dicarboxylic acid derivatives and subsequently to N-hydroxysuccinimide (NHS) ester [37] with slight modification. The polymer PLGA (62.5 µM) (MW-8000) was dissolved in 20 ml of dichloromethane (DCM) and 52 µM EDC was added in this solution. The resulting mixture was stirred overnight to obtain a clear yellow solution.
5.7µM of 5.0G PPI dendrimers was dissolved in 20.0 ml of methanol and in this solution activated PLGA solution was added. The resultant sample was stirred continuously 48 hours by using a magnetic stirrer in dark at room temperature. The final product was dialyzed by dialysis membrane (MWCO 12-14 kDa, Himedia, India) for 24 hours to remove free PLGA and unconjugated dendrimer. Excess solvent in this dialyzed product was removed by rotary vacuum evaporator (Scheme 1). Characterization of Dendro PLGA nanoconjugate was carried out by using FTIR, nuclear magnetic resonance (1H NMR) spectroscopy, and transmission electron microscopic (TEM) microscopy. 2.4. Drug loading and entrapment efficiency The known molar concentrations of PPI 5.0G dendrimer and DPNC were dissolved separately in phosphate buffer saline (PBS) (pH-7.4) and mixed with insulin solution in hydrochloride acid solution (0.1 M) which was also dissolved in same solvent. The mixed solutions were incubated with slow magnetic stirring (50 rpm) using teflon beads for 24 h at 2-80C. This preparation was taken and dialyzed in cellulose dialysis membrane (MWCO 12-14 kDa, Himedia, India) against PBS (pH7.4) under sink condition for 15 min to remove the unentrapped drug from the formulation, which was then estimated spectrophotometrically (λmax 562 nm) (UV 1601, Shimadzu, Japan)
by using bicinchoninic acid assay (BCA) kit to determine
indirectly the amount of loaded drug [38]. 2.5. In vitro drug release The in vitro drug release studies were carried out to determine the release of insulin from plain PPI dendrimer and DPNC by equilibrium dialysis (MWCO 12-14 kDa, Himedia, India) method [39]. Both drug loaded plain PPI dendrimer and DPNC (2 ml) were placed in the dialysis sacs separately. The sacs were hermetically sealed, and suspended in 30 ml of release medium PBS (pH-7.4), on magnetic stirrer
maintained at 370C. At predetermined time intervals; 1 ml of the release medium was withdrawn from both release assembly and this sample was used for the estimation of free insulin content. Every time, the amount of aliquot was removed and replaced by the equal amount of fresh PBS (pH-7.4) to maintain the sink condition during the experiment. The drug release was determined after appropriate dilution, using BCA kit at 562 nm. 2.6. Comparison of hemolytic toxicity of 5.0G PPI dendrimer and PLGA conjugated 5.0 G PPI dendrimer The RBC suspension was obtained as per the well-known and reported procedure for hemolytic studies [40,41]. In brief, the RBC suspension (5% hematocrit) of the human blood was collected in HiAnticlot blood collection vials (Himedia Labs, India). 0.5 ml of suitably diluted PLGA conjugated and nonconjugated PPI 5.0G were added to 4.5 ml of normal saline and incubated for 1hr with RBC suspension. This allowed comparison of the hemolysis data of the dendrimer and PLGA conjugated dendritic architectures to assess the effect of PLGA conjugation on hemolysis. After centrifugation, supernatants were taken and diluted with an equal volume of normal saline and absorbance was measured at 540 nm. RBC suspension was added to 5 ml of 0.9% NaCl solution (normal saline) and 5 ml distilled water, respectively to obtain 0 and 100% hemolysis. The degree of hemolysis was determined by the following equation:
ABs % Haemolysis = × 100 AB 100 where, ABs = Absorbance for the sample; and AB100 = Absorbance for control without formulation 2.7. Stability studies of 5.0 G PPI dendrimer and dendro PLGA nanoconjugate
Insulin loaded PPI dendrimer and dendro PLGA nanoconjugate as exposed to accelerated conditions of temperature and light. The formulation was taken in different vials and stored in dark (amber color vials) and in light (colorless vials) at 0ºC, room temperature and 50±2ºC in thermostatically controlled oven for a period of 5 weeks. The samples were analyzed every week for any precipitation, turbidity, crystallization, color change, consistency and drug leakage. The data obtained was used for the analysis of any physical and chemical degradation, the required storage conditions and the precautions required for storage. The samples were initially clear and transparent at 0ºC. The loss of drug from the formulation was ascertained after storage at accelerated conditions. The known amount of formulation was kept in benzoylated cellulose tubing (Sigma, USA) and dialyzed across the tubing. The external medium [PBS (pH-7.4)] was monitored for the content of the drugs spectrophotometrically. The percentage increase in drug release from the formulation was used to analyze the effects of accelerated conditions of storage on the formulations. Integrity of encapsulated drug was measured by SDS-PAGE, FTIR and CD spectroscopy. 2.8. In vivo studies The male albino rats (Sprague Dawley strain, 120±5 g) were used for in vivo experimental study. All the animal studies were conducted in accordance with the protocol approved by the Institutional Animal Ethical Committee of Dr. H. S. Gour University, Sagar (M.P.), India (Reg. No. 379/01/ab/CPCSEA). The animals were divided into 4 groups: Group 1 Control, Group 2 Plane insulin, Group 3 insulin loaded PPI dendrimer, Group 4 insulin loaded DPNC. Diabetes was induced in albino rats by single intraperitoneal injection of streptozocin (50 mg/kg). Both insulin loaded preparation DPNC, PPI dendrimer dissolved in PBS (pH-7.4) and plain insulin was also dissolved with PBS (pH-7.4) in the dose of 4 IU/kg body weight of rat and
administered through intravenous (IV) route in the albino rats of respective groups excluding control group. The control group was injected with PBS (pH-7.4). Blood glucose level of all animals was checked just before starting experiment by taking blood (0.1 mL) from the retro orbital plexus of the rats. Following the treatment, blood sample were collected from the retro orbital plexus of rats at predefined time intervals 0, 0.25, 0.5, 1, 2, 3, 4, 8, 12, 16 and 24 hr, and analyzed for glucose level in blood. 3. Results 3.1. Synthesis of PPI dendrimer Synthesis of 0.5G PPI was confirmed by IR peaks, mainly of nitrile at 2244.3 cm-1 and CH2 bending at 1467.7 cm-1 (Fig. 1a). All the nitrile terminal 0.5G PPI got converted into (NH2)4, which was confirmed by IR of PPI 1.0G that exhibited major peak at 3427.3 cm-1 of amine (N-H stretch) (Fig. 1b). Likewise, IR peaks also confirmed synthesis of 5.0G PPI dendrimers. The main peaks are of C-C bend (1106.6 cm-1); C-N stretch (1312.4 cm-1); C-H bend (1410.4 cm-1, 1463.2 cm-1); N-H deflection of amine (1665.5 cm-1) and primary amine at 3409.8 cm-1 (N-H stretch), confirming nitrile terminal groups of dendrimer were converted to amine terminals (Fig. 1c). The synthesis was also confirmed by proton NMR by the obtained major peaks and shifts. NMR of 5.0G PPI dendrimer revealed peaks of alkane were obtained between 0.6-1.25 ppm while peaks of alkyl amine were obtained between 1.3-1.9 ppm. Incompletely cyanoethyleted dendrimer was also evident between 3.2 to 3.8 ppm with integral value 37.86 (NCH2CH2-CN). Substituted alkyl amines exhibited peak between 3.2-3.8 ppm and primary amines exhibited peak at 7.99 ppm (Fig. 1d). TEM images of 5.0G PPI dendrimer proved their nanometric size (Fig. 1e). 3.2. Conjugation of PLGA to PPI dendrimer
The conjugation of PLGA to PPI dendrimer was confirmed by IR, NMR and TEM microscopy. The FTIR analysis of the conjugated system shows the characteristic peak of secondary amide at 3358.1 cm-1, with the additional peak of CH stretch at 2956.2 cm-1 and C=O stretch at 1667.0 cm-1 were observed (Fig. 2a). NMR spectroscopy results also prove the conjugation of PLGA with PPI dendrimer. Peak of ether linkage appears at 3.48-3.65 ppm, which proves the conjugation. The characteristic peak of amide linkage appeared near 7.27 ppm, which was absent in 5.0 G PPI dendrimer (Fig. 2b). The electron microscopic analysis of conjugated system displayed nanometric sized units, as evident from TEM. The increase in size of conjugated system also provides a strong evidence of the formation of DPNC (Fig. 2c). 3.3. Hemolytic toxicity The toxicity is due to the polycationic nature of the PPI dendrimers. However PLGA conjugation of dendrimers was found to decrease the hemolysis of the RBC considerably at all concentrations due to shielding or coating of the charged quaternary ammonium ion that is generally formed on the amine terminated whole generations of PPI dendrimers, responsible for hemolysis. The whole generation of amine-terminated charged 5.0G PPI dendrimers showed hemolytic toxicity 1.585±0.061, 2.181±0.063 and 3.443±0.036 at 0.01, 0.03 and 0.05 % w/v concentrations, respectively. However, the conjugation of PLGA to the dendrimers was found to have decreased the hemolysis of the RBCs significantly to 0.621±0.052, 1.145±0.0.065 and 1.660±0.083 at 0.01, 0.03 and 0.05 % w/v concentrations, respectively (Fig. 3). This was due to the inhibition of interaction of RBCs with the charged quaternary ammonium ion as determined by interaction with RBCs. These data can also favor the enhanced in vivo safety by our developed PLGA conjugated dendrimer.
3.4. Drug loading and entrapment efficiency The drug loading was performed by dialysis method using cellulose membrane (MWCO 12-14 kDa, Himedia, India) and un-entrapped drug was determined spectrophotometrically .The dendritic formulation was retained in the membrane due to its molecular weight and hyper branched polymeric spherical constitution while the free drug easily comes out of the membrane. The drug entrapment and retention is low in case of non conjugated dendrimer as compared to the conjugate system. This may be due to the covering of peripheral portion of dendrimer by PLGA polymer, which was responsible for the steric hindrance and as a consequence drug loading was improved. The entrapment efficiency of the system increased from PPI (16%) to DPNC (70%). 3.5. In vitro drug release The results of in vitro release of insulin from PPI dendrimer and DPNC are presented in Fig. 3a. The in vitro drug release profile shows that there is a slower release rate of insulin from DPNC as compared to PPI dendrimer. While the PPI completely releases the drug by 12 h, the PPI-based nanoconjugate, prolonged the release rate up to 36 h. This fact can once again be explained on the basis of steric hindrance at the periphery, which slows down and hence prolongs the release of insulin from DPNC as compared to PPI dendrimer (Fig. 4a). Once the therapeutic action has been achieved the conjugates may undergo renal clearance in fraction [42]. 3.6. Stability studies of PLGA conjugated dendrimer formulations The stability study carried out on the drug-PLGA conjugated dendrimer complexes at various accelerated conditions of temperature (0°C, RT, and 50°C) and light showed that the dendritic formulations are stable even at higher temperature if kept in dark (amber color vials) but are unstable in presence of light (colorless vials). There was change in color and precipitation noted after five weeks when kept at 50°C
in presence of light but no such change was observed at the same temperature in dark (Fig. 4b). However, there was drug loss from the formulation observed at higher temperature and it was found to be even greater in the presence of light. This may be due to higher reaction kinetics in presence of light at high temperature. The dendritic structures are supposed to be more open at higher temperature and this change in surface characteristics might cause the conformational changes in the structure and release of drug. SDS-PAGE of insulin showed that the drug retained native conformation after processing since the peak of insulin recovered from DPNC was matched with authentic sample of insulin (Fig. 5). Secondary structure of insulin after processing has been confirmed by FTIR spectroscopy (Fig. 6a). CD spectroscopy has been also carried out to further to confirm the preservation of secondary structure of insulin which is necessary for the protein therapeutic activity. CD spectrum of plain insulin showed 2 minima at 208 and 222 nm. These data was in close agreement with results presented by previous studies [43]. There was minor deviation in the negative maximum at about 220 nm for insulin recovered from DPNC. The secondary structure for insulin (native conformation) may have slightly changed after processing. The possible explanation for such observation would be the shear forces which acted during the formulation process, though not representing denaturation or loss of insulin activity (Fig. 6b). 3.7. In vivo studies Insulin loaded DPNC exhibited the longest time to decrease the blood glucose levels; while the insulin loaded PPI dendrimer exhibited the intermediate time, i.e. between insulin loaded DPNC and insulin suspension formulation, to decrease blood glucose level. Finally these results show that the insulin loaded DPNC exhibit the slower onset of glucose lowering over prolonged time period. This result also indicate
that DPNC stabilize the insulin during processing which is a major drawback associated with the market insulin formulation (Fig. 7). 4. Discussion Protein and peptides makes very important group of therapeutic agents but their instability during processing and storage damage their native structure which reduces their biological activity, inducing aggregation and render the proteins immunogenic, leading ultimately to precipitation [44]. The use of nanocarriers not only protects the proteins from different adverse conditions during storage but also act as a reservoir for control release of proteins. In present study, we synthesized a novel nanoconjugate system for the stabilization as well as delivery of protein. Dendrimers are a class of highly branched spherical polymeric systems. Two types of dendrimers, the polyamidoamine (PAMAM) [45] and PPI are commercially used [46]. Both types are highly soluble in aqueous solutions and have a unique surface covered by primary amino groups [47]. Due to their well-defined monodispersity, controlled architecture, nanosize (20-40 nm), and shape, they are often considered most promising vehicles for the stabilization and delivery of drugs, proteins, and other therapeutic agents [48-51]. Therefore, in a short time, they have emerged as the most promising drug delivery carriers. In this work, we conjugate the PLGA polymer to 5.0 G PPI dendrimer to form DPNC for the stabilization of protein. The drug entrapment and retention were low in case of non conjugated dendrimer as compared to the conjugate system. This might be due to the covering of peripheral portion of dendrimer by PLGA polymer, which was responsible for the steric hindrance and as a consequence drug loading was improved. The in vitro release of insulin from DPNC showed controlled release of drug as compared to insulin loaded unconjugated dendrimer in PBS (pH-7.4). This may be due to the
entrapment of insulin molecule in between long PLGA chain, possibly due to the electrostatic, hydrophobic interaction or hydrogen bonding. The hemolytic toxicity of the unconjugated PPI dendrimer as well as DPNC was carried out to determine the interaction between dendrimer and RBCs. The toxicity of the unconjugated PPI dendrimer is due to the polycationic nature of the PPI dendrimer. The modification of cationic dendrimers with PLGA shields the positive charge on the dendrimer surface as in case of PEGylation and leads to a decrease in cytotoxicity [52]. The stability study carried out on the insulin loaded PPI dendrimer and DPNC at various accelerated conditions of temperature (00C, RT, 500C) and light showed that the dendritic formulations were stable even at higher temperature if they are kept in dark (amber color vials), but unstable in the presence of light (colorless vials). The drug loss from the dendritic formulation at higher temperature and in presence of light was found to be more, as compared to room temperature and in dark, due to the destabilization and opening of dendritic structure, causing the loss of drug to a greater extent. Integrity of insulin recovered from DPNC confirmed by SDS-PAGE, FTIR and CD spectroscopy. Results obtained from these methods showed that the secondary structure for insulin (native conformation) retained after processing which are necessary for protein activity. In vivo results suggest that the insulin loaded DPNC exhibits slower onset of action and reduction in blood glucose level was found to be prolonged time period. These results also indicate that DPNC stabilizes the insulin during processing which is a major drawback associated with the market formulations of insulin. 5. Conclusions
Dendrimers are used as carrier macromolecules for many bioactives agents due to its targeting, imaging, and drug delivery property. When terminal amino group of PPI dendrimer are shielded by a polymer, then the toxicity of dendrimer is reduced and drug loading efficiency is also increased. The Dendro PLGA nanoconjugate retained the native structure of protein during processing which was confirmed by the SDS PAGE analysis, FTIR spectroscopy and CD spectroscopy. Non conjugated dendrimer showed more leakage of protein in adverse conditions as compared to the PLGA conjugated dendrimer. Furthermore the in vivo results of protein loaded DPNC also proved the retention of therapeutic activity of protein. Declaration of interest The authors report no conflicts of interest in this work. Acknowledgement Mr. Amit Kumar Tiwari acknowledges the All India Council of Technical Education, New Delhi, India for the grant of Junior Research Fellowship and Bicon Ltd., Banglore (India) for providing the gift sample of Human insulin. References [1] S.P. Schwendeman, M.A. Klibanov, R. Langer, M.R. Brandon. in: S. Cohen, H. Bernstein (Eds.), Microparticulate systems for the delivery of proteins and vaccines, Marcel Dekker, New York (1996) 1-49. [2] S.L. Nail. in: K. Park (Ed.), Controlled drug delivery challenges and strategies, American Chemical Society, Washington, DC (1997) 185-203. [3] Schoneich C, Hageman MJ, Borchardt RT. in: K. Park (Ed.), Controlled drug delivery challenges and strategies, American Chemical Society, Washington, DC (1997) 205-27. [4] J.L. Cleland, M.F. Powell, S.J. Shire. The development of stable protein formulations: a close look at protein aggregation, deamidation, and oxidation. Crit Rev Ther Drug Carr Syst 10 (1993) 307-77.
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Scheme 1. Schematic representation for conjugation of PPI 5.0G dendrimer to PLGA. Fig. 1. FTIR spectrum of (a) 0.5G (b) 1.0G (c) 5.0G PPI dendrimer; (d) NMR spectrum of 5.0G PPI dendrimer; (e) TEM photograph of 5.0G PPI dendrimer. Fig. 2. (a) FTIR (b) NMR spectrum (c) TEM photograph of PLGA conjugated dendrimer.
Fig. 3. Hemolytic toxicity profile of plain dendrimer and different PLGA-dendritic conjugates (n = 3). Fig. 4. (a) Cumulative percent drug release from dendritic formulations in PBS 7.4 as recipient medium (n = 3); (b) Percent drug leakage from drug loaded DPNC at different storage condition up to 5 weeks (n = 6) Fig. 5. SDS –PAGE after processing [Lane 1-molecular marker in kDa (180, 116, 58, 48.5, 36.5, 26.6, 10); Lane 2-Insulin; Lane 3-Insulin recovered from PPI dendrimer, Lane 4 – Insulin recovered from DPNC]. Fig. 6. (a) FTIR (b) CD-Spectra of insulin obtained from DPNC. Fig. 7. Blood glucose level in diabetic rats after administration of different formulations containing insulin equivalent to 4 IU/kg.
3_Graphical abstract .
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