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Catanionic nanocarriers as a potential vehicle for insulin delivery
Colloids and Surfaces B: Biointerfaces 188 (2020) 110759
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Catanionic nanocarriers as a potential vehicle for insulin delivery a,b,d,
c,d
Soledad Stagnoli *, Lucas Sosa Alderete , M. Alejandra Luna R. Dario Falconea,b, Ana M. Niebylskic,d, N. Mariano Correaa,b,*
a,b
T c,d
, Elizabeth Agostini ,
a
Instituto para el Desarrollo Agroindustrial y de la Salud (IDAS, UNRC-CONICET), Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, X5804ZAB, Río Cuarto, Córdoba, Argentina Departamento de Química. Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, X5804ZAB, Río Cuarto, Córdoba, Argentina c Instituto de Biotecnología Ambiental y Salud (INBIAS, UNRC-CONICET), Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, X5804ZAB, Río Cuarto, Córdoba, Argentina d Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, X5804ZAB, Río Cuarto, Córdoba, Argentina b
A R T I C LE I N FO
A B S T R A C T
Keywords: Catanionic surfactant Insulin Glycemia Vesicle
Diabetes is a disease that affects millions of people in the World, constituting a global problem. Patients are administered insulin subcutaneous injections, resulting in high costs and frequent infections in the injection site. A possible solution to this problem may be the use of nanotechnology. Nanotransporters can act as specific release systems able to overcome the current limitations to drug delivery. Liposomes and vesicles can deliver drugs directly and efficiently to the site of action, decreasing toxicity and adverse effects. In previous studies, we demonstrated the biocompatibility and safety of catanionic benzyl nhexadecyldimethylammonium 1,4 -bis-2-ethylhexylsulfosuccinate (BHD-AOT) vesicles using both in vitro and in vivo tests. Thus, the aims of this work were to evaluate the ability of the BHD-AOT vesicles to encapsulate insulin; to analyze the structural properties and stability of the system, vesicle-Insulin (VIn), at different pH conditions; and to study the ability of VIn to decrease the glycemia in miceby different administration routes. Our results showed that 2 and 5 mg mL−1 of vesicles were able to encapsulate about 55 % and 73 % of insulin, respectively. The system VIn showed a significant increase in size from 120 to 350 nm, changes in the surface zeta potential value, and high stability to different pH conditions. A significant decrease of the glycemia after VIn administration was demonstrated in in vivo assays, including the oral route. Our results reveal that BHD-AOT vesicles may be an appropriate system to encapsulate and protect insulin, and may be a potential system to be administrated in different ways as an alternative strategy to conventional therapy.
1. Introduction
specific release systems capable of overcoming the usual limitations to delivery [2]. They are called like this due to their nanometric-scale size (nm), which makes it easy for them to travel through the organism and reach the desired sites of release [3] such as tumors and diseased tissues. Nanotransporters can minimize the effects of the drug on surrounding tissues and, at the same time, maximize the action of the drug on the affected organ or tissue [3], as well as improve the stability of hydrophobic drugs, protect the drug from unfavorable conditions within the organism, enhance drug biodistribution, and reduce secondary effects and toxicity [4]. In the last few years, much attention has been paid to vesicles and liposomes [4]. They are organized systems that deliver drugs directly and efficiently to the site of action, with reduced toxicity and adverse
One of the main limitations of drug delivery is its systemic and nonspecific distribution, which decreases the efficiency and bioavailability to cells or target tissues (i.e. a deficiency in specific tropism). The drug may also be unable, or find it difficult to cross the selectively permeable membrane due to highly specific molecular mechanisms. In order to obtain effective drug concentrations in the target cell, it is necessary to administer relatively high doses that usually lead to unwanted toxicological and immunological effects [1]. One alternative is to encapsulate bioactive compounds within biocompatible transporters that allow the effective entry of active agents into the target cell [1]. Nanotransporters, in particular, can act as
⁎ Corresponding authors at: Instituto para el Desarrollo Agroindustrial y de la Salud (IDAS, UNRC-CONICET), Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, X5804ZAB, Río Cuarto, Córdoba, Argentina. E-mail addresses: [email protected] (S. Stagnoli), [email protected] (N.M. Correa).
https://doi.org/10.1016/j.colsurfb.2019.110759 Received 22 September 2019; Received in revised form 27 November 2019; Accepted 23 December 2019 Available online 24 December 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
Colloids and Surfaces B: Biointerfaces 188 (2020) 110759
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due to its implication in more than twenty diseases [18], among them, Alzheimer's, Parkinson's, diabetes in Huntington's disease and type 2 diabetes [19]. Currently, increases in diabetes in all regions of the world have been demonstrated. Diabetes has severe complications, such as kidney problems, peripheral arterial and cardiovascular diseases and blindness, triggering direct effects on the quality of life of the patients and high demand of health services and high economic costs [20]. To control the glucose levels in bood at normal levels, a daily subcutaneous injection of insulin is essential for the treatment of diabetic patients. Although this traditional treatment can control the glucose levels in the blood to the normal level, the significant problem is that the traditional administration might result in a series of consequences, such as high costs and poor patient compliance in addition to side effects, like injection site infections [21]. Insulin, an anabolic hormone, is usually administered subcutaneously and has a medium-short lifespan (30 min) after administration [22], which may cause a hypoglycemic shock. However, this is the only route currently available [23,24]. Thus, efforts to develop oral delivery systems have been made. However, the oral bioavailability of protein/peptide drugs is mainly poor in humans due to enzymatic inactivation and absorption barriers present in the gastrointestinal tract (GT) [25]. The latter include physical barriers like viscous mucous layers and tight junctions of enterocytes in the GT, chemical barriers of low stomach pH, and biological barriers, such as enzymatic degradation. Therefore, overcoming these problems is essential for enhancing absorption of administered peptide-based drugs. Moreover, the development of a delivery vehicle for oral administration of insulin, which can deliver and release insulin in a self-regulated way, presents an interesting challenge [25] as a new alternative strategy for the treatment of diabetes mellitus. The aims of this work were to i) evaluate the ability of the catanionic BHD-AOT vesicles to encapsulate insulin; ii) to analyze the structural properties and stability of the system, vesicle-Insulin (VIn) at different pH conditions and; iii) to study the ability of VIn system to decrease the glycemia, in mice, through different administration routes.
effects [5–7]. Moreover, their therapeutic use is advantageous, since they enhance drug bioavailability, especially when using drugs that are mostly insoluble in aqueous media [8]. There have been reports that these colloidal systems can upgrade drug solubility and pharmacokinetic properties [9], being able to encapsulate both hydrophobic drugs in their lipidic bilayer, and hydrophilic drugs in their aqueous core [9]. Those studies have also shown the highest compatibility with biological systems [10]. All of these characteristics give their use and application to numerous advantages over other nanotransporters [9]. Recently geat strides have been made in the development of drugs incorporated into vesicles and liposomes for the treatment of eye and skin afflictions [11]. Currently, there are a number of products in the market that use these systems as nanotransporters [9]. Vesicles have been used to transport enzymes, antibiotics, and antimicrobial and antifungal compounds [4]. Likewise, there has been progress in their use as nanotransporters of anticancerous and antitumoral drugs [9,12]. These products have been entering the market in the last few years, but they still need to overcome a set of obstacles for their application to be widespread [13]. Their low stability during long storage periods is among such obstacles, as well as the tendency of phospholipids to become oxidized under certain physiological conditions, as is the case with intestinal and gastric fluids [13]. The efficiency of conventional liposomes is compromised by their instability in the gastrointestinal tract and their poor permeability through the epithelial membrane [14]. Their bilayer is usually modified so as to counter these complications [13] but such modifications may make it difficult and costly to produce and use liposomes on an industrial scale [9]. Taking all of this into consideration, there have been recent advances in the development of vesicles formed by molecules of a different type than conventional phospholipids [15]. In this sense, we obtained a catanionic system through the synthesis of a novel surfactant, benzyl n-hexadecyldimethylammonium 1,4 -bis-2-ethylhexylsulfosuccinate (BHDAOT (Scheme 1)), resulting from the combination of anionic sodium 1,4-bis (2-ethylhexyl) sulfosuccinate (Na-AOT) and cationic benzyldimethylhexadecylammonium chloride (BHDC), whose counterions were eliminated [16]. When dissolved in water, this surfactant spontaneously creates unilamellar vesicles, without the addition of energy to the system, thus offering an advantage for drug delivery over traditional vesicles formed by phospholipids [17]. In previous studies, we demonstrated in vitro, that these vesicles are biocompatible at concentrations lower than 0.05 mg mL−1. Moreover, no differences were found in enzymatic activity of transaminases, phosphatase and lactate dehydrogenase, as well as in the behavior of mice injected with the different doses of vesicles (3.4 and 13.8 mg/kg) between control and treatment group during 30 days. Also, these vesicles were resistant to acid pH (2) for 90 min [16]. These results suggest that the BHD-AOT vesicles are innocuousness in vitro (≤0.05 mg mL−1) and in vivo (≤13.8 mg/kg) studies and, therefore, they are promising alternatives for drug delivery including the oral route, due to its potential stability in similar conditions of gastrointestinal system, its biocompatibility, and its innocuousness in biological systems [16]. Another complication is the aggregation of bioactive compounds receiving attention in the fields of biology, medicine and biophysics,
2. Experimental 2.1. BHD-AOT vesicles formation The benzyl n-hexadecyldimethylammonium 1,4 -bis-2-ethylhexylsulfosuccinate, BHD-AOT, surfactant was obtained using an equimolar mixture of sodium 1,4-bis (2-ethylhexyl) sulfosuccinate (NaAOT) and benzyl-n-hexadecyldimethylammonium chloride (BHDC), both from Sigma (> 99 % purity). To obtain the vesicles, BHD-AOT surfactant was hydrated [16,17]. A stock solution of BHD-AOT vesicles (5 mg mL−1) was prepared by weighting an appropriated amount of BHD-AOT and, diluting with aqueous solution to obtain the desired concentration. An opalescent solution was obtained by hand shaking for two minutes at room temperature. The formation of the BHD-AOT vesicles was confirmed by Dynamic Light Scattering (DLS) technique, a similar diameter was obtained, as previously reported [17]. All samples were prepared and used immediately after of their formation. 2.2. BHD-AOT vesicles /insulin complex (VIn) formation An insulin solution (1 mg mL−1) was prepared in distillated water at pH 7, and the BHD-AOT vesicles/insulin complex was formed (Supplementary Material) [15,26]. To detect the insulin incorporation into the vesicles, different assays were performed: partition constants (Kp) of insulin, detection of insulin incorporated into the vesicles, and encapsulation efficiency. To evaluate the physicochemical properties of the VIn complex, the following experiments were performed: evaluation of size and surface charges, analysis of surface morphology, structural and morphological stability at different pH values, evaluation of insulin integrity at different pH
Scheme 1. Molecular structure of the catanionic surfactant BHD-AOT. 2
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values, evaluation of insulin release and in vitro stability at different pH values. To evaluate the biological activity of VIn complex, analysis of in vivo glycemia level was determined afterwards. (Supplementary Material). 3. Results and discussion 3.1. Partition constant (Kp) for insulin To investigate whether insulin could be incorporated into the BHDAOT bilayer, an estimation of the insulin Kp value was performed. To this, changes in insulin emission intensity at different BHD-AOT vesicles concentrations, at λem =305 nm (λexc =276 nm) was collected and plotted (Fig. S1).
Scheme 2. Schematic representation of the possible location site of insulin within the unilamellar catanionic vesicle bilayer.
are electrically stabilized, while nanocarriers with zeta potential intermediate values (between 30 mV and -30 mV) tend towards coagulation [32,33]. We suggest that the high surface potential of our vesicles with or without insulin increases the repulsive forces preventing the aggregation and maintaining the stability and monodispersity (low polydispersity of 0.2) of the vesicles with and without the hormone. It is important to remark that the VIn complex remains stable and monodisperse in sizy at pH 7 at 37 °C, even after seventy-five days of its preparation. This feature turns our VIn complex in an interesting candidate for drug delivery.
3.2. Detection of insulin into the vesicles by PAGE technique To confirm whether insulin was encapsulated by the vesicles, an electrophoresis assay was performed. In order to determine the ability of our BHD-AOT vesicles to encapsulated insulin, two different surfactant concentrations were tested (2 and 5 mg mL−1) using the same insulin concentration (2 mg mL−1) (Fig. S2). 3.3. Encapsulation efficiency of insulin by vesicles Table S1 shows the percentages of insulin encapsulation using different BHD-AOT concentrations. According to the equation S4, the efficiencies to encapsulate insulin were 55 % and 73 % at 2 mg mL−1 and 5 mg mL−1 BHD-AOT, respectively.
3.5. Studies of surface morphology of VIn complex Vesicles without insulin showed a smooth surface undulation, with an approximate size of 188 nm and a thickness of 35 nm (Fig. S3 A) while the vesicles with insulin showed a rough and irregular surface, with an approximate size of 384 nm and a thickness of 170 nm (Fig. S3 B). (See Supplementary Material).
3.4. Determination of size and surface charges (Z potential, ζ) of the VIn complex The first assays consisted of studies to determine structural changes, which will be the consequence of a possible interaction of the vesicles with insulin. An initial evaluation of changes in the sizes and surface charges (zeta potential) of the system was observed after the hormone incorporation. Table S2 shows that the vesicles prepared with insulin significantly increased their sizes from 120 to 350 nm. Additionally, the electronegative charge of the catanionic vesicles bilayer decreased from -34.15 to -28.52 mV. The increase in the size of the vesicles could be associated with the insertion of insulin at the bilayer of the vesicles. Tah et al. [15] recorded the same behavior in catanionic vesicles of SDS-CTAB, where a significant increase in the size of the vesicles from 200 nm to 500 nm was shown, accompanied by a change in the zeta potential (ζ), from −32 mV to −28 mV [15]. The authors have attributed these changes to the insulin incorporation into the bilayer. BHD-AOT vesicles with the incorporation of insulin decreased the negative charge of the bilayer. Although, it is known that the surface potential of insulin in aqueous solution pH 7 is −11.8 mV [15], in this case, the fact that the incorporation of the hormone (electronegative) does not increase the electrical charge of the vesicles, suggests that insulin, when it interacts with the vesicles, exists at the bilayer without exposing the charged groups to the surface. The probable insulin location within the vesicles bilayer was shown in Scheme 2. The ζ value is an indicator of system stability, associated with the behavior of the membrane and, its interaction with the medium in terms of repulsion between adjacent charged particles in solution [27]. For supramolecular systems, such vesicles, a high zeta potential value indicates stability; in other words, the solution or dispersion will resist aggregation [28–30]. When the potential decreases, particles attract each other and overcome repulsion, producing coagulation instead of dispersion [30,29]. This feature in biological media such as blood could avoid the interaction with the plasma proteins [31]. Therefore, nanocarriers with zeta potential higher than 30 mV or lower than −30 mV
3.6. Analyses of size and stability of the VIn at different pH values In order to analyze the structural stability of VIn, the diameter (Fig. S4 A) and polydispersity (Fig. S4 B) values were evaluated by DLS technique. The incorporation of insulin into the interface, as well as the VIn complex stability, were evaluated under different pH conditions (pH 2, 7 and 8) at 37 °C. The pH 2 and 37 °C were used in order to imitate the physiological situation regarding the gastric pH and the body temperature. pH 7 and 8 were used in order to simulate the pH of biological fluids, such as blood and the intestinal fluid, respectively. (Refer to the Supplementary Material). 3.7. Morphological analyses of VIn complex at different pH values As observed in Fig. 1, the vesicles maintained their morphology with slight changes at the surface level when the insulin was incorporated at different pH (Fig. 1B–D). In addition, the typical formations of amyloid fibrils as a consequence of denaturation of insulin, by break of the vesicles, were not observed in any of the pH analyzed [34]. These Figures (A–D) also confirm the results of the DLS regarding the stability of the system VIn when exposed to different pH conditions (2, 7 and 8) for 90 min. 3.8. Analyses of insulin structure at different pH values Circular dichroism constitutes an important tool in the chemistry and structural biology of proteins [35]. The native structure of insulin can be altered by changes in medium pH, as is the case with gastrointestinal fluids, by the action of gastrointestinal enzymes, or by physical factors that limit its intestinal absorption. As shown in Fig. 2, the dichroism spectra of free insulin (denatured 3
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Fig. 1. TEM images. A) BHD-AOT vesicles (2 mg mL−1) without insulin (control) at pH 7; B) BHD-AOT vesicles with insulin at (2 mg mL−1) 0.5 mg mL−1 at pH 7; C) BHD-AOT vesicles (2 mg mL−1) with insulin at 0.5 mg mL−1 at pH 2; D) BHD-AOT vesicle (2 mg mL−1) with insulin at 0.5 mg mL−1 at pH 8 for 90 min. All assays were performed at 37 °C.
structure. 3.9. Analysis of insulin released and in vitro stability at different pH values Changes in the fluorescence intensity of FI and EI were evaluated at λem=305 nm, simulating the pH of the gastrointestinal tract (stomach, intestine) and the blood tissue. Fluorescence emission remained constant in time at 0.5 mg mL−1 insulin concentration, at pH 7 and 37 °C. However, when the same solution was exposed to pH 2 and pH 8, the fluorescence emissions of insulin were considerably higher and lower than the control, respectively (Fig. 3A). Fig. 3B showed that the fluorescence of insulin associated to VIn complex at different pH values (2, 7, 8) at 37 °C did not change in time (0−150 min). After VIn complex disruption with Triton X-100, insulin was exposed to the medium displaying brusque changes in fluorescence emission intensity, similar to those observed in Fig. 3A. Therefore, according to these results, the vesicle not only protects the insulin as was mentioned above, but also avoids its passive releasing. In a low pH solution, conformational changes allowed an alternative aggregation that leads to the formation of insoluble amyloid fibers [39], causing denaturation of the insulin [40–42]. These changes involve the folding of the hormone, exposing the tryptophan to the medium, increasing the intensity of fluorescence emission [19]. This increase in fluorescence emission has been widely used as an indicator of the degree of amyloid formation [19]. In agreement with all of this, and considering that the insulin concentration of vesicles is the same in the three pH values investigated, we observed that when insulin was exposed to acidity conditions, fluorescence emission increased, likely as a consequence of protein folding, fibrillation, and denaturation. Whittingham et al. [43] suggested that acid conditions increase molecular vulnerability to conformational change and dissociation. It is critical to understand protein aggregation in a variety of biomedical situations ranging from conformational disorders (such as Alzheimer's disease) [44] to the production, stability, and administration of pharmaceutical products based on proteins such as insulin [44]. Administering insulin to a diabetic patient can result in the formation of an amyloid structure at the site of injection, which leads to hormone instability, and the creation of an insoluble and
Fig. 2. Circular dichroism spectra of: non-denatured insulin (0.5 mg mL−1) at pH 7, denatured insulin (0.5 mg mL−1) and insulin (0.5 mg mL−1) associated to vesicle (2 mg mL−1) at different pH values (2, 7 and 8) at 90 min.
and non-denatured) and the insulin associated to the VIn complex at different pH (2, 7, 8) were obtained. The non-denatured insulin and the insulin associated to the vesicles showed a valley at 208 nm and a shoulder around 223 nm. This dichroic spectrum coincided with data reported in literature with a typical secondary structure (α-helix conformation) with predominance of biological activity of the insulin [36–38]. The spectrum of insulin associated with vesicles showed almost no changes in the negative and positive band positions, with only a slight decrease in the maximum intensity, with respect to non-denatured insulin. On the other hand, the secondary structure of the denatured insulin was considerably altered in agreement with previous research [15,36]. These results show that the vesicles do not generate any structural alteration of the hormone and, in addition, protects it from the extreme conditions of the medium, preventing a potential process of fibrillation and denaturation, while preserving its physiologically functional native 4
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Fig. 4. Blood glucose levels before vesicle administration (basal) and at different times (15, 30,60, 90 and 120 min) after the VIn administration. Control (filled square ◼), VIn intraperitoneal (IP, empty circles ○), subcutaneous (SC,filled rhombus ♦) and oral (empty triangles∇). a, b, c, d p < 0.05 vs IP basal values; e, f, g, h p < 0.005 vs SC basal values, j = p < 0.05 vs oral basal values. Error bars report standard deviation of six independent measurements (n = 6). Variance of repeated measures (MANOVA) and Duncan's a-posteriori test as a post hoc test were used.
(p = 0.06) and statistically significant at 120 min (p = 0.0113) of administration with respect to their corresponding baseline value. In the case of control mice, changes in glycemic level at the evaluated times and in relation to its baseline value were not observed. Blood glucose level was reduced by approximately 67 % (p = 0.000184) in the subcutaneous route and 70 % (p = 0.00012) in intraperitoneal via at 90 min post administration. The oral treatment reduced the blood glucose levels by 30 and 40 % at 90 and 120 min respectively. This result can be explained because the subcutaneous and intraperitoneal routes are direct pathways, which allow a faster arrival of the hormone to the systemic circulation, while the oral route has many physical and chemical barriers and a longer time of permanence of the complex in the gastrointestinal system [45]. It is important to note that the control group mice showed a high and unchanged glucose level (180 mg/dL) at all analyzed times. On the other hand, the basal levels of glycemia obtained before the corresponding administration of VIn (treatment) or V (control), were higher than those published in the literature (120 mg/dL). A possible explanation of this result could be due to the stress generated by the manipulation of the animals, a stimulus that generates the activation of the hypothalamus/ hypophysis / adrenal axis and sympathetic activation of the mice [46]. The activation of these axes promotes the release of hormones such as glucocorticoids, and adrenaline, which are responsible for the increase of glycemia due to their glycogenolytic and glyconeogenic activity [46]. Even though this assay is the first biological test about the activity of VIn complex in vivo, the results obtained are novel, with great biological importance. The encapsulated insulin into the vesicles could avoid the fibrillation when this hormone was administrated by subcutaneous and intraperitoneal routes, diminishing the administration problems that might affect glycemic control in patients with diabetes, making it an important therapeutic consideration [47,48]. In this context, we propose our catanionic vesicles as protector agents to avoid these administration troubles by different routes. On the other hand, because the VIn complex decreases the glucose levels in blood in all the treated mice after 90 or 120 min of oral administration, it can be thought that the vesicles have the ability to transport efficiently the insulin across the gastrointestinal tract, overcoming all biological, physical and chemical barriers. According to these results, we suggest that the catanionic vesicles are able to interact with the enterocyte cells releasing the insulin into the cell, to then
Fig. 3. Fluorescence emission intensity at λem=305 nm of A) 0.5 mg mL−1 insulin B) insulin (0.5 mg mL−1) associated to vesicle (2 mg mL−1). All assays were performed at 37 °C exposed to different pH values (2, 7 and 8) for different times (0, 30, 60, 90, 120 and 150 min). In B, after 150 min TritonX-100 was added to disrupt VIn complex and release insulin. Circles (○), square (◼) and triangles (▲) represents pH 2, 7 and 8, respectively. Error bars report standard deviation of three independent measurements. λex=280 nm.
biologically inactive complex. This pathology is known as insulin-derived amyloidosis [19]. The results suggest that BHD-AOT vesicles protected and did not allow to release the hormone in the time, avoiding the folding and reassembly (fibrillation) of the insulin (Fig. 3B) since no changes were observed in the fluorescence intensity under different conditions while the insulin was inside of the vesicles. When the VIn complex was destroyed, the hormone was exposed to the medium (pH) conditions, showing a behavior similar to that observed in Fig. 3A and confirming the protective role of the vesicles on the integrity of the protein. 3.10. In vivo experiments This assay was performed to evaluate the biological activity of the encapsulated insulin, and the capacity to overcome different barriers using several route administrations (intraperitoneal, subcutaneous and oral). As shown in Fig. 4, the blood glucose levels of the control and treated mice were analyzed over time. The ANOVA test showed the effect of the treatment vs time (p = 0.00001) and Duncan's a-posteriori test indicated that there are no significant differences between the baseline values of any group. Mice that received intraperitoneal and/or subcutaneous administrations of the VIn complex showed a significant decrease of the blood glucose levels up to 15 min in relation to their corresponding baseline values. On the other hand, the mice that received an oral administration of the VIn complex, although, presenting a decrease in glycemia over time, this decrease was at 90 min 5
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References
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Liu, Z. Zhang, Y. Ding, J. Lv, R. Ma, Y. An, L. Shi, A. Glucose and H2O2 dual-sensitive nanogels for enhanced glucose-responsive insulin delivery, Nanoscale 11 (2019) 9163–9917. [22] D.R. Owens, Clinical pharmacology of human insulin, Diabetes Care 16 (1993) 90–100. [23] R.B. Shah, F. Ahsan, M.A. Khan, Oral delivery of proteins: progress and prognostication, Crit. Rev. Ther. Drug Carrier Syst. 19 (2002) 135–169. [24] M. Morishita, N.A. Peppas, Is the oral route possible for peptide and protein drug delivery, Drug Discov. 11 (2006) 905–910. [25] K. Suzuki, K.S. Kim, Y.H. Bae, Long-term oral administration of Exendin-4 to control type 2 diabetes in a rat model, J. Control. Release 294 (2019) 259–267. [26] S. Park, S.G. Choi, E. Davaa, J. Park, Encapsulation enhancement and stabilization of insulin in cationic liposomes, Int. J. Pharm. 415 (2011) 267–272. [27] S. Choi, S.W. Kim, Controlled release of insulin from injectable biodegradable triblock copolymer depot in ZDF rats, Pharm. 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4. Conclusion The high stability of the BHD-AOT catanionic vesicles were verified under different parameters, such as temperature, mainly in the range of 37 °C, and upon acidic and basic pH conditions. These characteristics will make them more efficient to transport different drugs like insulin than the conventional liposomes. Regarding the evaluation of the system as an insulin nanocarrier, it was possible to conclude that all the results obtained allowed to verify the potential of the system to transport high quantities of insulin through different administration routes, including the oral one. We have obtained an effective vehicle to overcome all the barriers of the different routes, including the gastrointestinal one, to reach the target cells and finally modify the glycemia of an organism. It is important to remark the protective role provided by vesicles to a peptide drug, such as insulin. This work opens a new venue to investigate and evaluate the capacity and efficient of the system to transport different pharmaceutical drugs, mainly in oral administration, as a new alternative therapy to replace the conventional therapies. CRediT authorship contribution statement Soledad Stagnoli: Conceptualization, Methodology, Formal analysis, Writing - original draft, Visualization. Lucas Sosa Alderete: Resources. M. Alejandra Luna: Conceptualization, Methodology, Formal analysis. Elizabeth Agostini: Resources. R. Dario Falcone: Conceptualization, Methodology, Formal analysis, Writing - original draft, Visualization. Ana M. Niebylski: Conceptualization, Methodology, Formal analysis, Writing - original draft, Supervision. N. Mariano Correa: Conceptualization, Methodology, Writing - review & editing, Visualization, Supervision, Project administration, Funding acquisition. Declaration of Competing Interest There are no conflicts of interest to declare. Acknowledgments Financial support from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP CONICET 112-201101-00204 and 112-2015-0100283), Universidad Nacional de Río Cuarto, Agencia Nacional de Promoción Científica y Técnica (PICT-2015-2151 and PICT 2015-0585), Ministerio de Ciencia y Tecnología de Córdoba (PID 2018) is gratefully acknowledged. We also acknowledge the technical assistance of Miguel Bueno (Bioterium) and Claudia Nomode Docampo (Electronic Microscopy), and Florencia Sgarlatta and Porf. Iliena Martinez for English language correction. N.M.C, R.D.F., L.S.A, E.A hold a research position at CONICET. A.S.S. thanks CONICET for a research scholarship. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110759. 6
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L.F. Nazar, The importance of nanometric passivating films on cathodes for Li-Air batteries, ACS Nano 8 (2014) 12483–12493. L. Xu, L. Feng, R. Dong, J. Hao, S. Dong, Transfection efficiency of DNA enhanced by association with salt-free catanionic vesicles, Biomacromolecules 14 (2013) 2781–2789. I.B. Bekard, D.E. Dunstan, Tyrosine autofluorescence as a measure of bovine insulin fibrillation, Biophys. J. 97 (2009) 2521–2531. M. Ishtikhar, M.V. Khan, S. Khan, S.K. Chaturvedi, Journal of Biomolecular Structure and Dynamics Biophysical and molecular docking insight into interaction mechanism and thermal stability of human serum albumin isoforms with a semisynthetic water-soluble camptothecin analog irinotecan hydrochloride, J. Biomol. Struct. Dyn. 34 (2016) 1545–1560. M. Niu, Y. Lu, L. Hovgaard, Liposomes containing glycocholate as potential oral insulin delivery systems: preparation, in vitro characterization, and improved protection against enzymatic degradation, Int. J. Nanomedicine 6 (2011) 1155–1166. W. Tiyaboonchai, J. Woiszwillo, R.C. Sims, C.R. Middaugh, Insulin containing polyethylenimine – dextran sulfate nanoparticles, Int. J. Pharm. 255 (2003) 139–151. M. Chinisaz, A. Ebrahim-habibi, A. Dehpour, P. Yaghmaei, Structure and function of anhydride-modi fi ed forms of human insulin: in silico, in vitro and in vivo studies, Eur. J. Pharm. Sci. 96 (2017) 342–350. J.B. Wang, Y.M. Wang, C.M. Zeng, Quercetin inhibits amyloid fibrillation of bovine insulin and destabilizes preformed fibrils, Biochem. Biophys. Res. Commun. 415 (2011) 675–679.
[40] J. Jayamani, G. Shanmugam, Gallic acid, one of the components in many plant tissues, is a potential inhibitor for insulin amyloid fibril formation, Eur. J. Med. Chem. 85 (2014) 352–358. [41] K. Kitagawa, Y. Misumi, M. Ueda, Y. Hayashi, M. Tasaki, K. Obayashi, T. Yamashita, H. Jono, H. Arima, Y. Ando, Inhibition of insulin amyloid fibril formation by cyclodextrins, Amyloid. 22 (2015) 181–186. [42] L. Nielsen, S. Frokjaer, J. Brange, V.N. Uversky, A.L. Fink, Probing the mechanism of insulin fibril formation with insulin mutants, Biochemistry 40 (2001) 8397–8409. [43] J.L. Whittingham, D.J. Scott, K. Chance, A. Wilson, J. Finch, J. Brange, G. Guy Dodson, Insulin at pH 2: Structural analysis of the conditions promoting insulin fibre formation, J. Mol. Biol. 318 (2002) 479–490. [44] M. Stefani, Biochemical and biophysical features of both oligomer/fibril and cell membrane in amyloid cytotoxicity, FEBS J. 277 (2010) 4602–4613. [45] M. Chen, K. Sonaje, K. Chen, H. Sung, Biomaterials A review of the prospects for polymeric nanoparticle platforms in oral insulin delivery, Biomaterials 32 (2011) 9826–9838. [46] J. Axelrod, T.D. Reisine, Stress hormones: their interaction and regulation, Science 224 (1984) 452–459. [47] M.R. Nilsson, Insulin amyloid at injection sites of patients with diabetes, Amyloid 23 (2016) 139–147. [48] F.E. Dische, C. Wernstedt, G.T. Westermark, P. Westermark, M.B. Pepys, J.A. Rennie, S.G. Gilbey, P.J. Watkins, Insulin as an amyloid-fibril protein at sites of repeated insulin injections in a diabetic patient, Diabetologia 31 (1988) 158–161.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Catanionic nanocarriers as a potential vehicle for insulin delivery a,b,d,
c,d
Soledad Stagnoli *, Lucas Sosa Alderete , M. Alejandra Luna R. Dario Falconea,b, Ana M. Niebylskic,d, N. Mariano Correaa,b,*
a,b
T c,d
, Elizabeth Agostini ,
a
Instituto para el Desarrollo Agroindustrial y de la Salud (IDAS, UNRC-CONICET), Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, X5804ZAB, Río Cuarto, Córdoba, Argentina Departamento de Química. Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, X5804ZAB, Río Cuarto, Córdoba, Argentina c Instituto de Biotecnología Ambiental y Salud (INBIAS, UNRC-CONICET), Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, X5804ZAB, Río Cuarto, Córdoba, Argentina d Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, X5804ZAB, Río Cuarto, Córdoba, Argentina b
A R T I C LE I N FO
A B S T R A C T
Keywords: Catanionic surfactant Insulin Glycemia Vesicle
Diabetes is a disease that affects millions of people in the World, constituting a global problem. Patients are administered insulin subcutaneous injections, resulting in high costs and frequent infections in the injection site. A possible solution to this problem may be the use of nanotechnology. Nanotransporters can act as specific release systems able to overcome the current limitations to drug delivery. Liposomes and vesicles can deliver drugs directly and efficiently to the site of action, decreasing toxicity and adverse effects. In previous studies, we demonstrated the biocompatibility and safety of catanionic benzyl nhexadecyldimethylammonium 1,4 -bis-2-ethylhexylsulfosuccinate (BHD-AOT) vesicles using both in vitro and in vivo tests. Thus, the aims of this work were to evaluate the ability of the BHD-AOT vesicles to encapsulate insulin; to analyze the structural properties and stability of the system, vesicle-Insulin (VIn), at different pH conditions; and to study the ability of VIn to decrease the glycemia in miceby different administration routes. Our results showed that 2 and 5 mg mL−1 of vesicles were able to encapsulate about 55 % and 73 % of insulin, respectively. The system VIn showed a significant increase in size from 120 to 350 nm, changes in the surface zeta potential value, and high stability to different pH conditions. A significant decrease of the glycemia after VIn administration was demonstrated in in vivo assays, including the oral route. Our results reveal that BHD-AOT vesicles may be an appropriate system to encapsulate and protect insulin, and may be a potential system to be administrated in different ways as an alternative strategy to conventional therapy.
1. Introduction
specific release systems capable of overcoming the usual limitations to delivery [2]. They are called like this due to their nanometric-scale size (nm), which makes it easy for them to travel through the organism and reach the desired sites of release [3] such as tumors and diseased tissues. Nanotransporters can minimize the effects of the drug on surrounding tissues and, at the same time, maximize the action of the drug on the affected organ or tissue [3], as well as improve the stability of hydrophobic drugs, protect the drug from unfavorable conditions within the organism, enhance drug biodistribution, and reduce secondary effects and toxicity [4]. In the last few years, much attention has been paid to vesicles and liposomes [4]. They are organized systems that deliver drugs directly and efficiently to the site of action, with reduced toxicity and adverse
One of the main limitations of drug delivery is its systemic and nonspecific distribution, which decreases the efficiency and bioavailability to cells or target tissues (i.e. a deficiency in specific tropism). The drug may also be unable, or find it difficult to cross the selectively permeable membrane due to highly specific molecular mechanisms. In order to obtain effective drug concentrations in the target cell, it is necessary to administer relatively high doses that usually lead to unwanted toxicological and immunological effects [1]. One alternative is to encapsulate bioactive compounds within biocompatible transporters that allow the effective entry of active agents into the target cell [1]. Nanotransporters, in particular, can act as
⁎ Corresponding authors at: Instituto para el Desarrollo Agroindustrial y de la Salud (IDAS, UNRC-CONICET), Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, X5804ZAB, Río Cuarto, Córdoba, Argentina. E-mail addresses: [email protected] (S. Stagnoli), [email protected] (N.M. Correa).
https://doi.org/10.1016/j.colsurfb.2019.110759 Received 22 September 2019; Received in revised form 27 November 2019; Accepted 23 December 2019 Available online 24 December 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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due to its implication in more than twenty diseases [18], among them, Alzheimer's, Parkinson's, diabetes in Huntington's disease and type 2 diabetes [19]. Currently, increases in diabetes in all regions of the world have been demonstrated. Diabetes has severe complications, such as kidney problems, peripheral arterial and cardiovascular diseases and blindness, triggering direct effects on the quality of life of the patients and high demand of health services and high economic costs [20]. To control the glucose levels in bood at normal levels, a daily subcutaneous injection of insulin is essential for the treatment of diabetic patients. Although this traditional treatment can control the glucose levels in the blood to the normal level, the significant problem is that the traditional administration might result in a series of consequences, such as high costs and poor patient compliance in addition to side effects, like injection site infections [21]. Insulin, an anabolic hormone, is usually administered subcutaneously and has a medium-short lifespan (30 min) after administration [22], which may cause a hypoglycemic shock. However, this is the only route currently available [23,24]. Thus, efforts to develop oral delivery systems have been made. However, the oral bioavailability of protein/peptide drugs is mainly poor in humans due to enzymatic inactivation and absorption barriers present in the gastrointestinal tract (GT) [25]. The latter include physical barriers like viscous mucous layers and tight junctions of enterocytes in the GT, chemical barriers of low stomach pH, and biological barriers, such as enzymatic degradation. Therefore, overcoming these problems is essential for enhancing absorption of administered peptide-based drugs. Moreover, the development of a delivery vehicle for oral administration of insulin, which can deliver and release insulin in a self-regulated way, presents an interesting challenge [25] as a new alternative strategy for the treatment of diabetes mellitus. The aims of this work were to i) evaluate the ability of the catanionic BHD-AOT vesicles to encapsulate insulin; ii) to analyze the structural properties and stability of the system, vesicle-Insulin (VIn) at different pH conditions and; iii) to study the ability of VIn system to decrease the glycemia, in mice, through different administration routes.
effects [5–7]. Moreover, their therapeutic use is advantageous, since they enhance drug bioavailability, especially when using drugs that are mostly insoluble in aqueous media [8]. There have been reports that these colloidal systems can upgrade drug solubility and pharmacokinetic properties [9], being able to encapsulate both hydrophobic drugs in their lipidic bilayer, and hydrophilic drugs in their aqueous core [9]. Those studies have also shown the highest compatibility with biological systems [10]. All of these characteristics give their use and application to numerous advantages over other nanotransporters [9]. Recently geat strides have been made in the development of drugs incorporated into vesicles and liposomes for the treatment of eye and skin afflictions [11]. Currently, there are a number of products in the market that use these systems as nanotransporters [9]. Vesicles have been used to transport enzymes, antibiotics, and antimicrobial and antifungal compounds [4]. Likewise, there has been progress in their use as nanotransporters of anticancerous and antitumoral drugs [9,12]. These products have been entering the market in the last few years, but they still need to overcome a set of obstacles for their application to be widespread [13]. Their low stability during long storage periods is among such obstacles, as well as the tendency of phospholipids to become oxidized under certain physiological conditions, as is the case with intestinal and gastric fluids [13]. The efficiency of conventional liposomes is compromised by their instability in the gastrointestinal tract and their poor permeability through the epithelial membrane [14]. Their bilayer is usually modified so as to counter these complications [13] but such modifications may make it difficult and costly to produce and use liposomes on an industrial scale [9]. Taking all of this into consideration, there have been recent advances in the development of vesicles formed by molecules of a different type than conventional phospholipids [15]. In this sense, we obtained a catanionic system through the synthesis of a novel surfactant, benzyl n-hexadecyldimethylammonium 1,4 -bis-2-ethylhexylsulfosuccinate (BHDAOT (Scheme 1)), resulting from the combination of anionic sodium 1,4-bis (2-ethylhexyl) sulfosuccinate (Na-AOT) and cationic benzyldimethylhexadecylammonium chloride (BHDC), whose counterions were eliminated [16]. When dissolved in water, this surfactant spontaneously creates unilamellar vesicles, without the addition of energy to the system, thus offering an advantage for drug delivery over traditional vesicles formed by phospholipids [17]. In previous studies, we demonstrated in vitro, that these vesicles are biocompatible at concentrations lower than 0.05 mg mL−1. Moreover, no differences were found in enzymatic activity of transaminases, phosphatase and lactate dehydrogenase, as well as in the behavior of mice injected with the different doses of vesicles (3.4 and 13.8 mg/kg) between control and treatment group during 30 days. Also, these vesicles were resistant to acid pH (2) for 90 min [16]. These results suggest that the BHD-AOT vesicles are innocuousness in vitro (≤0.05 mg mL−1) and in vivo (≤13.8 mg/kg) studies and, therefore, they are promising alternatives for drug delivery including the oral route, due to its potential stability in similar conditions of gastrointestinal system, its biocompatibility, and its innocuousness in biological systems [16]. Another complication is the aggregation of bioactive compounds receiving attention in the fields of biology, medicine and biophysics,
2. Experimental 2.1. BHD-AOT vesicles formation The benzyl n-hexadecyldimethylammonium 1,4 -bis-2-ethylhexylsulfosuccinate, BHD-AOT, surfactant was obtained using an equimolar mixture of sodium 1,4-bis (2-ethylhexyl) sulfosuccinate (NaAOT) and benzyl-n-hexadecyldimethylammonium chloride (BHDC), both from Sigma (> 99 % purity). To obtain the vesicles, BHD-AOT surfactant was hydrated [16,17]. A stock solution of BHD-AOT vesicles (5 mg mL−1) was prepared by weighting an appropriated amount of BHD-AOT and, diluting with aqueous solution to obtain the desired concentration. An opalescent solution was obtained by hand shaking for two minutes at room temperature. The formation of the BHD-AOT vesicles was confirmed by Dynamic Light Scattering (DLS) technique, a similar diameter was obtained, as previously reported [17]. All samples were prepared and used immediately after of their formation. 2.2. BHD-AOT vesicles /insulin complex (VIn) formation An insulin solution (1 mg mL−1) was prepared in distillated water at pH 7, and the BHD-AOT vesicles/insulin complex was formed (Supplementary Material) [15,26]. To detect the insulin incorporation into the vesicles, different assays were performed: partition constants (Kp) of insulin, detection of insulin incorporated into the vesicles, and encapsulation efficiency. To evaluate the physicochemical properties of the VIn complex, the following experiments were performed: evaluation of size and surface charges, analysis of surface morphology, structural and morphological stability at different pH values, evaluation of insulin integrity at different pH
Scheme 1. Molecular structure of the catanionic surfactant BHD-AOT. 2
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values, evaluation of insulin release and in vitro stability at different pH values. To evaluate the biological activity of VIn complex, analysis of in vivo glycemia level was determined afterwards. (Supplementary Material). 3. Results and discussion 3.1. Partition constant (Kp) for insulin To investigate whether insulin could be incorporated into the BHDAOT bilayer, an estimation of the insulin Kp value was performed. To this, changes in insulin emission intensity at different BHD-AOT vesicles concentrations, at λem =305 nm (λexc =276 nm) was collected and plotted (Fig. S1).
Scheme 2. Schematic representation of the possible location site of insulin within the unilamellar catanionic vesicle bilayer.
are electrically stabilized, while nanocarriers with zeta potential intermediate values (between 30 mV and -30 mV) tend towards coagulation [32,33]. We suggest that the high surface potential of our vesicles with or without insulin increases the repulsive forces preventing the aggregation and maintaining the stability and monodispersity (low polydispersity of 0.2) of the vesicles with and without the hormone. It is important to remark that the VIn complex remains stable and monodisperse in sizy at pH 7 at 37 °C, even after seventy-five days of its preparation. This feature turns our VIn complex in an interesting candidate for drug delivery.
3.2. Detection of insulin into the vesicles by PAGE technique To confirm whether insulin was encapsulated by the vesicles, an electrophoresis assay was performed. In order to determine the ability of our BHD-AOT vesicles to encapsulated insulin, two different surfactant concentrations were tested (2 and 5 mg mL−1) using the same insulin concentration (2 mg mL−1) (Fig. S2). 3.3. Encapsulation efficiency of insulin by vesicles Table S1 shows the percentages of insulin encapsulation using different BHD-AOT concentrations. According to the equation S4, the efficiencies to encapsulate insulin were 55 % and 73 % at 2 mg mL−1 and 5 mg mL−1 BHD-AOT, respectively.
3.5. Studies of surface morphology of VIn complex Vesicles without insulin showed a smooth surface undulation, with an approximate size of 188 nm and a thickness of 35 nm (Fig. S3 A) while the vesicles with insulin showed a rough and irregular surface, with an approximate size of 384 nm and a thickness of 170 nm (Fig. S3 B). (See Supplementary Material).
3.4. Determination of size and surface charges (Z potential, ζ) of the VIn complex The first assays consisted of studies to determine structural changes, which will be the consequence of a possible interaction of the vesicles with insulin. An initial evaluation of changes in the sizes and surface charges (zeta potential) of the system was observed after the hormone incorporation. Table S2 shows that the vesicles prepared with insulin significantly increased their sizes from 120 to 350 nm. Additionally, the electronegative charge of the catanionic vesicles bilayer decreased from -34.15 to -28.52 mV. The increase in the size of the vesicles could be associated with the insertion of insulin at the bilayer of the vesicles. Tah et al. [15] recorded the same behavior in catanionic vesicles of SDS-CTAB, where a significant increase in the size of the vesicles from 200 nm to 500 nm was shown, accompanied by a change in the zeta potential (ζ), from −32 mV to −28 mV [15]. The authors have attributed these changes to the insulin incorporation into the bilayer. BHD-AOT vesicles with the incorporation of insulin decreased the negative charge of the bilayer. Although, it is known that the surface potential of insulin in aqueous solution pH 7 is −11.8 mV [15], in this case, the fact that the incorporation of the hormone (electronegative) does not increase the electrical charge of the vesicles, suggests that insulin, when it interacts with the vesicles, exists at the bilayer without exposing the charged groups to the surface. The probable insulin location within the vesicles bilayer was shown in Scheme 2. The ζ value is an indicator of system stability, associated with the behavior of the membrane and, its interaction with the medium in terms of repulsion between adjacent charged particles in solution [27]. For supramolecular systems, such vesicles, a high zeta potential value indicates stability; in other words, the solution or dispersion will resist aggregation [28–30]. When the potential decreases, particles attract each other and overcome repulsion, producing coagulation instead of dispersion [30,29]. This feature in biological media such as blood could avoid the interaction with the plasma proteins [31]. Therefore, nanocarriers with zeta potential higher than 30 mV or lower than −30 mV
3.6. Analyses of size and stability of the VIn at different pH values In order to analyze the structural stability of VIn, the diameter (Fig. S4 A) and polydispersity (Fig. S4 B) values were evaluated by DLS technique. The incorporation of insulin into the interface, as well as the VIn complex stability, were evaluated under different pH conditions (pH 2, 7 and 8) at 37 °C. The pH 2 and 37 °C were used in order to imitate the physiological situation regarding the gastric pH and the body temperature. pH 7 and 8 were used in order to simulate the pH of biological fluids, such as blood and the intestinal fluid, respectively. (Refer to the Supplementary Material). 3.7. Morphological analyses of VIn complex at different pH values As observed in Fig. 1, the vesicles maintained their morphology with slight changes at the surface level when the insulin was incorporated at different pH (Fig. 1B–D). In addition, the typical formations of amyloid fibrils as a consequence of denaturation of insulin, by break of the vesicles, were not observed in any of the pH analyzed [34]. These Figures (A–D) also confirm the results of the DLS regarding the stability of the system VIn when exposed to different pH conditions (2, 7 and 8) for 90 min. 3.8. Analyses of insulin structure at different pH values Circular dichroism constitutes an important tool in the chemistry and structural biology of proteins [35]. The native structure of insulin can be altered by changes in medium pH, as is the case with gastrointestinal fluids, by the action of gastrointestinal enzymes, or by physical factors that limit its intestinal absorption. As shown in Fig. 2, the dichroism spectra of free insulin (denatured 3
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Fig. 1. TEM images. A) BHD-AOT vesicles (2 mg mL−1) without insulin (control) at pH 7; B) BHD-AOT vesicles with insulin at (2 mg mL−1) 0.5 mg mL−1 at pH 7; C) BHD-AOT vesicles (2 mg mL−1) with insulin at 0.5 mg mL−1 at pH 2; D) BHD-AOT vesicle (2 mg mL−1) with insulin at 0.5 mg mL−1 at pH 8 for 90 min. All assays were performed at 37 °C.
structure. 3.9. Analysis of insulin released and in vitro stability at different pH values Changes in the fluorescence intensity of FI and EI were evaluated at λem=305 nm, simulating the pH of the gastrointestinal tract (stomach, intestine) and the blood tissue. Fluorescence emission remained constant in time at 0.5 mg mL−1 insulin concentration, at pH 7 and 37 °C. However, when the same solution was exposed to pH 2 and pH 8, the fluorescence emissions of insulin were considerably higher and lower than the control, respectively (Fig. 3A). Fig. 3B showed that the fluorescence of insulin associated to VIn complex at different pH values (2, 7, 8) at 37 °C did not change in time (0−150 min). After VIn complex disruption with Triton X-100, insulin was exposed to the medium displaying brusque changes in fluorescence emission intensity, similar to those observed in Fig. 3A. Therefore, according to these results, the vesicle not only protects the insulin as was mentioned above, but also avoids its passive releasing. In a low pH solution, conformational changes allowed an alternative aggregation that leads to the formation of insoluble amyloid fibers [39], causing denaturation of the insulin [40–42]. These changes involve the folding of the hormone, exposing the tryptophan to the medium, increasing the intensity of fluorescence emission [19]. This increase in fluorescence emission has been widely used as an indicator of the degree of amyloid formation [19]. In agreement with all of this, and considering that the insulin concentration of vesicles is the same in the three pH values investigated, we observed that when insulin was exposed to acidity conditions, fluorescence emission increased, likely as a consequence of protein folding, fibrillation, and denaturation. Whittingham et al. [43] suggested that acid conditions increase molecular vulnerability to conformational change and dissociation. It is critical to understand protein aggregation in a variety of biomedical situations ranging from conformational disorders (such as Alzheimer's disease) [44] to the production, stability, and administration of pharmaceutical products based on proteins such as insulin [44]. Administering insulin to a diabetic patient can result in the formation of an amyloid structure at the site of injection, which leads to hormone instability, and the creation of an insoluble and
Fig. 2. Circular dichroism spectra of: non-denatured insulin (0.5 mg mL−1) at pH 7, denatured insulin (0.5 mg mL−1) and insulin (0.5 mg mL−1) associated to vesicle (2 mg mL−1) at different pH values (2, 7 and 8) at 90 min.
and non-denatured) and the insulin associated to the VIn complex at different pH (2, 7, 8) were obtained. The non-denatured insulin and the insulin associated to the vesicles showed a valley at 208 nm and a shoulder around 223 nm. This dichroic spectrum coincided with data reported in literature with a typical secondary structure (α-helix conformation) with predominance of biological activity of the insulin [36–38]. The spectrum of insulin associated with vesicles showed almost no changes in the negative and positive band positions, with only a slight decrease in the maximum intensity, with respect to non-denatured insulin. On the other hand, the secondary structure of the denatured insulin was considerably altered in agreement with previous research [15,36]. These results show that the vesicles do not generate any structural alteration of the hormone and, in addition, protects it from the extreme conditions of the medium, preventing a potential process of fibrillation and denaturation, while preserving its physiologically functional native 4
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Fig. 4. Blood glucose levels before vesicle administration (basal) and at different times (15, 30,60, 90 and 120 min) after the VIn administration. Control (filled square ◼), VIn intraperitoneal (IP, empty circles ○), subcutaneous (SC,filled rhombus ♦) and oral (empty triangles∇). a, b, c, d p < 0.05 vs IP basal values; e, f, g, h p < 0.005 vs SC basal values, j = p < 0.05 vs oral basal values. Error bars report standard deviation of six independent measurements (n = 6). Variance of repeated measures (MANOVA) and Duncan's a-posteriori test as a post hoc test were used.
(p = 0.06) and statistically significant at 120 min (p = 0.0113) of administration with respect to their corresponding baseline value. In the case of control mice, changes in glycemic level at the evaluated times and in relation to its baseline value were not observed. Blood glucose level was reduced by approximately 67 % (p = 0.000184) in the subcutaneous route and 70 % (p = 0.00012) in intraperitoneal via at 90 min post administration. The oral treatment reduced the blood glucose levels by 30 and 40 % at 90 and 120 min respectively. This result can be explained because the subcutaneous and intraperitoneal routes are direct pathways, which allow a faster arrival of the hormone to the systemic circulation, while the oral route has many physical and chemical barriers and a longer time of permanence of the complex in the gastrointestinal system [45]. It is important to note that the control group mice showed a high and unchanged glucose level (180 mg/dL) at all analyzed times. On the other hand, the basal levels of glycemia obtained before the corresponding administration of VIn (treatment) or V (control), were higher than those published in the literature (120 mg/dL). A possible explanation of this result could be due to the stress generated by the manipulation of the animals, a stimulus that generates the activation of the hypothalamus/ hypophysis / adrenal axis and sympathetic activation of the mice [46]. The activation of these axes promotes the release of hormones such as glucocorticoids, and adrenaline, which are responsible for the increase of glycemia due to their glycogenolytic and glyconeogenic activity [46]. Even though this assay is the first biological test about the activity of VIn complex in vivo, the results obtained are novel, with great biological importance. The encapsulated insulin into the vesicles could avoid the fibrillation when this hormone was administrated by subcutaneous and intraperitoneal routes, diminishing the administration problems that might affect glycemic control in patients with diabetes, making it an important therapeutic consideration [47,48]. In this context, we propose our catanionic vesicles as protector agents to avoid these administration troubles by different routes. On the other hand, because the VIn complex decreases the glucose levels in blood in all the treated mice after 90 or 120 min of oral administration, it can be thought that the vesicles have the ability to transport efficiently the insulin across the gastrointestinal tract, overcoming all biological, physical and chemical barriers. According to these results, we suggest that the catanionic vesicles are able to interact with the enterocyte cells releasing the insulin into the cell, to then
Fig. 3. Fluorescence emission intensity at λem=305 nm of A) 0.5 mg mL−1 insulin B) insulin (0.5 mg mL−1) associated to vesicle (2 mg mL−1). All assays were performed at 37 °C exposed to different pH values (2, 7 and 8) for different times (0, 30, 60, 90, 120 and 150 min). In B, after 150 min TritonX-100 was added to disrupt VIn complex and release insulin. Circles (○), square (◼) and triangles (▲) represents pH 2, 7 and 8, respectively. Error bars report standard deviation of three independent measurements. λex=280 nm.
biologically inactive complex. This pathology is known as insulin-derived amyloidosis [19]. The results suggest that BHD-AOT vesicles protected and did not allow to release the hormone in the time, avoiding the folding and reassembly (fibrillation) of the insulin (Fig. 3B) since no changes were observed in the fluorescence intensity under different conditions while the insulin was inside of the vesicles. When the VIn complex was destroyed, the hormone was exposed to the medium (pH) conditions, showing a behavior similar to that observed in Fig. 3A and confirming the protective role of the vesicles on the integrity of the protein. 3.10. In vivo experiments This assay was performed to evaluate the biological activity of the encapsulated insulin, and the capacity to overcome different barriers using several route administrations (intraperitoneal, subcutaneous and oral). As shown in Fig. 4, the blood glucose levels of the control and treated mice were analyzed over time. The ANOVA test showed the effect of the treatment vs time (p = 0.00001) and Duncan's a-posteriori test indicated that there are no significant differences between the baseline values of any group. Mice that received intraperitoneal and/or subcutaneous administrations of the VIn complex showed a significant decrease of the blood glucose levels up to 15 min in relation to their corresponding baseline values. On the other hand, the mice that received an oral administration of the VIn complex, although, presenting a decrease in glycemia over time, this decrease was at 90 min 5
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References
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4. Conclusion The high stability of the BHD-AOT catanionic vesicles were verified under different parameters, such as temperature, mainly in the range of 37 °C, and upon acidic and basic pH conditions. These characteristics will make them more efficient to transport different drugs like insulin than the conventional liposomes. Regarding the evaluation of the system as an insulin nanocarrier, it was possible to conclude that all the results obtained allowed to verify the potential of the system to transport high quantities of insulin through different administration routes, including the oral one. We have obtained an effective vehicle to overcome all the barriers of the different routes, including the gastrointestinal one, to reach the target cells and finally modify the glycemia of an organism. It is important to remark the protective role provided by vesicles to a peptide drug, such as insulin. This work opens a new venue to investigate and evaluate the capacity and efficient of the system to transport different pharmaceutical drugs, mainly in oral administration, as a new alternative therapy to replace the conventional therapies. CRediT authorship contribution statement Soledad Stagnoli: Conceptualization, Methodology, Formal analysis, Writing - original draft, Visualization. Lucas Sosa Alderete: Resources. M. Alejandra Luna: Conceptualization, Methodology, Formal analysis. Elizabeth Agostini: Resources. R. Dario Falcone: Conceptualization, Methodology, Formal analysis, Writing - original draft, Visualization. Ana M. Niebylski: Conceptualization, Methodology, Formal analysis, Writing - original draft, Supervision. N. Mariano Correa: Conceptualization, Methodology, Writing - review & editing, Visualization, Supervision, Project administration, Funding acquisition. Declaration of Competing Interest There are no conflicts of interest to declare. Acknowledgments Financial support from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP CONICET 112-201101-00204 and 112-2015-0100283), Universidad Nacional de Río Cuarto, Agencia Nacional de Promoción Científica y Técnica (PICT-2015-2151 and PICT 2015-0585), Ministerio de Ciencia y Tecnología de Córdoba (PID 2018) is gratefully acknowledged. We also acknowledge the technical assistance of Miguel Bueno (Bioterium) and Claudia Nomode Docampo (Electronic Microscopy), and Florencia Sgarlatta and Porf. Iliena Martinez for English language correction. N.M.C, R.D.F., L.S.A, E.A hold a research position at CONICET. A.S.S. thanks CONICET for a research scholarship. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110759. 6
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