Water-soluble cationic poly(β-cyclodextrin-co-guanidine) as a controlled vitamin B₂ delivery carrier

Abstract

Vitamin B₂ (VB₂, as an important nutrient) is effectively incorporated into novel water-soluble cationic β-cyclodextrin (β-CD) polymers in order to improve its physiochemical properties. The properties of encapsulated VB₂ are characterized by FT-IR, ¹H NMR and ¹³C NMR for interaction between host and guest; DSC for thermogram properties; DLS for size and size distribution; zeta potential for surface charges and host–guest interaction types and water solubility study. The proposed novel water-soluble cationic poly(β-CD-co-guanidine) (CAβCDP) is synthesized by β-CD and guanidine (GN) as a building block and epichlorohydrin (EP) as a crosslinker through a one-step polymerization procedure at varying molar ratios of GN related to EP and β-CD. These prepared polymers are characterized by FT-IR, ¹H NMR, ¹³C NMR, TGA, DTG, DSC, DDSC and Kjeldahl methods. In addition, the solubility of the CAβCDP in water media, in order to recognize the effect of entered GN in the solubility behaviour of CAβCDP, is determined. The in vitro release behavior of VB₂ from CAβCDPs is investigated in various simulated physiological media, and exhibited initial burst and then slow VB₂ release. It is found that the cationic β-CD polymers with high GN content exhibit slower vitamin release compared to the others.

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Introduction

VB₂ (Fig. 1), also known as 7,8-dimethyl-10-(10-D-ribityl)isoalloxazine or riboflavin, is one of the B complex family members, and is an essential vitamin for normal human health.¹ It is expected that homeostatic changes of VB₂ induce negative health outcomes, such as damaging changes in the nervous system, anemia, cataracts and growth retardation.² This vitamin has a key role in the oxidation–reduction of carbohydrates, lipids, amino acid metabolism and certain vitamins (vitamin B₆ and folate). It is the precursor of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), important coenzymes that are essential for cellular metabolism, proliferation, growth and survival.³ The other roles of this vitamin are anti-oxidant,^4–6 anti-inflammatory properties⁷ and immune normal performance role^8,9 that have stated in more recent studies. It seems that VB₂ has a protective role in the important tissues of the human body from ischemia resulting of oxidative injury¹⁰ and a role in both leukaemia and prostate cancer cells by diminish survival signalling. A numerous circumstances such as diabetes mellitus, chronic alcoholism,^11,12 Inflammatory Bowel Disease (IBD),^13,14 inborn errors metabolism of this vitamin like BVVL (infantile Brown–Vialetto–Van Laere) and Fazio Londe syndromes^15–18 are examples in which deficiency or suboptimal levels of this vitamin occurs. Optimizing body homeostasis of VB₂ is effective in management of disease such as in BVVL patients,^15–18 as well as in B₂-responsive multiple acyl-CoA dehydrogenase deficiency patients.^19,20 Humans and other mammals cannot synthesize the VB₂ endogenously, so they should receive this vitamin exogenously from food source typically.²¹ Chemically, the VB₂ compound is slightly water soluble and a highly sensitive to the light.^22,23 Biological and pharmacological activity of VB₂ can be reduced owing to its photo degradation and poor water solubility.^24–27 The development of foods fortified with VB₂ is a suitable strategy to protect people at risk of the VB₂ deficiency, to prevent and treat some different disorders,^28,29 but, application of this method is not easy due to the yellowish colour of this vitamin and its capacity to produce photosensitized reactions.³⁰ There have been performed various methods to improve photostability and solubility of VB₂.

Fig. 1 Chemical structure of VB₂.

Nowadays, encapsulation technology is growing to protect the enclosed substances from the surrounding environment, to increase their efficiency and to achieve their controlled release.^31,32 Numerous encapsulating compounds such as polymers, vesicles, micelles and hydrogels have been proposed in the most studies recently.^31–33 Among them, β-CDs are one of the most interested compound in this regard.^33,34

The β-CD is the macrocyclic oligosaccharide that is composed of D-glucose units join to each other by α-1,4-glucosidic linkages. The shape of β-CD molecule is similar to truncated cone with a hydrophobic interior cavity, including the glycosidic oxygens and methine protons, and the hydrophilic exterior surface, formed by secondary and primary hydroxyls on the large and small rims. Owing to its unique cavity, β-CD can easily form stable host–guest inclusion complexes with certain molecules in which they fit into this cavity with high selectivity. The main driving forces behind these complexes formation are hydrophobic and van der Waals interactions. Therefore, due to the polarity difference between the internal and external surfaces, bioactivity and physicochemical properties (stability, solubility, odor, etc.) of the encapsulated guest can be noticeably improved. For the capabilities to form inclusion complexes, low price, readily available and the presence of various reactive groups on the backbone chain, β-CDs are widely used as food additives in the food industry, for stabilization of flavors and elimination of undesired tastes.³⁵ In addition, β-CDs have frequently been applied in pharmacy, separation processes, analytical sciences and catalysis, as well as in the textile, cosmetic and packaging industry.^36–40 The application of β-CD in food industry is limited by its rather low aqueous solubility. To overcome these limitations, numerous chemical modification have been developed on β-CD molecule.

There have been numerous studies on the preparation of water-soluble derivatives of β-CD molecules, which two special categories of these derivatives are charged β-CDs and β-CD polymers. In order to combine both the favorable properties of charged β-CDs and β-CD polymers, a series of ionic β-CD polymers were prepared using various polyfunctional crosslinking agents.^41–49 Depending on the type of crosslinking agent is used during its polymerization some properties of β-CD, such as its stability to heat and pH, shear forces and its solubility could be modified. Among all the proposed crosslinkers, EP has been the most extensively utilized bifunctional crosslinking agent in the production of neutralized β-CD polymer due to its facile synthesis, high sorption properties and particular selectivity. On the other hand, existence of ionic crosslinkers in β-CD polymers structure have been shown increased loading ability toward different guest due to the cooperative action between the β-CD cavities and the surrounding ionic groups.^50,51 For this purpose, a series of cationic and anionic β-CD polymers were prepared by using various polyfunctional crosslinking agents. The mainly cationic β-CD polymers were synthesized by crosslinking β-CD with EP in the presence of choline chloride.^52,53 These cationic β-CD polymers have been applied in gene delivery,^54–57 in drug delivery systems as hydrogels and nanoparticles^58,59 and as drug carriers⁵² or more particular in glucose-sensitive multilayer films.^60,61 On the other hand, anionic polymers were prepared from crosslinking reaction between citric acid and β-CD.^36,62 These polymers promoted complexion capacities toward the numerous drugs⁶³ when they were immobilized onto biomaterial devices, for example vascular prostheses,^64,65 porous hydroxyapatite bone substitutes,⁶⁶ periodontal membranes⁶⁷ and visceral meshes.^68–70

In the last decades, different research groups decided to encapsulation of VB₂ with CDs. In this regards, inclusion complexes between β-CD,^71–75 α-CD⁷² and some of its derivatives such as 2-hydroxypropyl-β-CD (HP-β-CD)⁷⁶ with VB₂ have been reported. These researches have demonstrated that VB₂ can form stable inclusion complexes (molar ratio 1 : 1) with β-CD. Enter partial hydrophobic of VB₂ into the macrocyclic cavity causes enhancement of aqueous solubility of the encapsulated vitamin. But, the presence of ribityl substituent in VB₂ prevents the deep penetration of this compound into β-CD cavity.

Based on the mentioned above, owing to the significance of cationic polymers and limitation of loading VB₂ by α- and β-CDs, we synthesised a novel water-soluble cationic β-CD polymers, as a drug delivery carrier, by insertion of GN in structure of NEβCDP. The cationic polymer structure was characterized and their water solubility was measured. Then the VB₂ (as the model vitamin) was loaded in cationic polymers and some properties of the obtained complexes were analyzed. In the last step, vitamin-release behavior was also systematically evaluated.

Experimental section

Materials

β-CD was purchased from SDFCL, Mumbai. Epichlorohydrin, vitamin B₂, bromocresol green, methyl-red and membrane dialysis tube (benzoylated, molecular weight cut-off of 2000 Da) were purchased from Sigma-Aldrich Company. Guanidine hydrochloride, boric acid, potassium sulfate (K₂SO₄), copper(II) sulfate (Cu₂SO₄), sulfuric acid, hydrochloric acid (HCl), acetone and ethanol were purchased from Merck Company. Sodium hydroxide (NaOH) was purchased from Mojallali Company, Iran and neutral βCD polymer (NEβCDP) was synthesized as previously described.⁷⁷ Deionized (DI) water has been used in all experiment processes.

Synthesis and characterization of polymers

Synthesis of CAβCDPs. The mixture of β-CD and GN solution in the molar ratio of 1 : 5, 1 : 10 and 1 : 15 were prepared as follows: initially, β-CD solution was prepared by dissolving 5 g of β-CD in 8 mL of NaOH solution (33% w/w) and then stirred mechanically at an ambient temperature for an overnight. Then, calculated GN powder was added to the β-CD solution. The 3.5 mL of EP was rapidly added to the mixture. The temperature was monitored during polymerization and kept at 30 °C for 3 h and 50 min. This reaction was stopped by acetone addition. Then acetone was removed and the aqueous solution neutralized by addition of HCl 6 N. This solution kept at 50 °C overnight. The prepared CAβCDP solution was precipitated using ethanol and the obtained solid washed with ethanol and acetone for several times. The CAβCDP resultant was placed in the air at overnight and then dried in oven at 70 °C.

Determination of GN content on CAβCDP. The nitrogen content of CAβCDPs was determined by Kjeldahl method. About 0.5 g of CAβCDPs was first digested (digital digestion unit, PECO) by sulfuric acid in the presence of Cu₂SO₄ and K₂SO₄. After digestion, ammonia was distilled (distillation unit, PDU-500, PECO) into boric acid solution (4% w/w) and titrated with 0.2 N HCl using bromocresol green/methyl-red as a pH indicator.⁷⁸ The nitrogen content was calculated by the following formula:
T: the HCl solution volume (mL) used in CAβCDP titration; B: the HCl solution volume (mL) used in NEβCDP titration; C: the standard HCl solution concentration (mol L⁻¹); w: the testing sample weight (mg).

The GN content in CAβCDPs structure (Table 1) has been calculated as follows:

Table 1 The GN content and the yield of CAβCDPs prepared by different molar ratios of GN

	NEβCDP	CAβCDP-5	CAβCDP-10	CAβCDP-15
β-CD : EP : GN	1 : 15 : 0	1 : 15 : 5	1 : 15 : 10	1 : 15 : 15
Yield (%)	68	71	74	82
GN contents (%)	0	6.9 ± 1.6	14 ± 1.8	30.3 ± 2.3

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FT-IR spectrum analysis. The Fourier-transform infrared (FT-IR) (Bruker Tensor 27) spectra were measured at an ambient temperature in the range of 4000–400 cm⁻¹ using the potassium bromide pellet technique.

NMR spectroscopy. Nuclear magnetic resonance (NMR) spectra were conducted using a Varian Unity 400 spectrometer operated at 400 MHz for proton nuclear (¹H NMR) and at 100 MHz for carbon nuclear (¹³C NMR). Deuterated DMSO was used as a solvent, without an internal standard.

Thermal analysis. Differential scanning calorimetry (DSC), thermogravimetric measurements (TGA) and their derivatives were acquired on a Netzsch (STA 409 PG, Germany) instrument. About 15 mg of samples heated from 50 °C to 500 °C at a heating rate of 10 °C min⁻¹ under nitrogen atmosphere (flow rate of 50 mL min⁻¹).

Aqueous solubilities of β-CD polymers. Excess amounts of polymer samples were added to 2 mL of water, to ensure it is saturated. The solution was mechanically shaken for 4 h and then incubated overnight at ambient temperature. The solution was then filtered through a sintered glass funnel (Pyrex®). The filtrated polymers were dried in an oven for sufficient period until a constant weight being reached. The solubility was estimated from the difference between the initial amount and the residual amount of polymers.

Preparation and characterization of CAβCDP/VB₂ complexes

Preparation of CAβCDP/VB₂ complexes. The CAβCDPs/VB₂ complexes were prepared by using a co-precipitation method. A 300 mg of CAβCDP was dissolved in 50 mL of water, then, 6 mg of VB₂ dissolved in 50 mL water was slowly added to the solution with continuous agitation. The final solution was maintained for 48 h at ambient temperature until evaporation all of the solvent. The precipitate was washed with ethanol to clear VB₂ that absorbed on the surface of β-CD polymer and then it dried in a vacuum oven at 40 °C for 4 h until the weight kept constant. These processes are performed in darkness in order to protect VB₂ from photodegradation.

Solid-state characterization. Solid-state characterization of CAβCDPs/VB₂ were performed using FT-IR, ¹H NMR, ¹³C NMR and DSC analyses, by the same methods which have been described in the characterization of polymer section.

Aqueous solubilities of complexes. The solubility of CAβCDP/VB₂ complexes in water were conducted following the conditions described previously.

Dynamic light scattering (DLS) and zeta potential. The particle size and zeta potential of the resulting complexes were obtained using the Zetasizer Nano Series (ZEN 3600, Malvern Instruments Ltd., UK). These studies were directed on aqueous solutions of samples at a constant temperature (25 ± 0.1 °C). Zeta average size and zeta potential were calculated from the auto-correlated function using the DTS Software (version 5.02) from Malvern Instruments.

In vitro release studies

In order to carry out the in vitro release study, buffer solutions of pH 1.2, 7.4 and 10, were prepared. The VB₂ incorporated carriers (30 mg) were poured in 3 mL of aqueous buffered solution. The mixture was conducted into a membrane dialysis bag and then the bag was closed and transferred into a flask containing 30 mL of buffer solutions of desired pH at the physiological temperature (37 °C). The release media was stirred gently at a constant speed of 50 rpm and a sample solution (3 mL) was withdrawn at regular selected intervals and replaced with equal volumes of the fresh buffer. The amount of released vitamin was determined by measuring the absorbance of these solutions spectrophotometrically at 368, 360, and 361 nm for pH 1.2, 7.4 and 10, respectively. The amount of vitamin released was computed by comparison of the solution absorbance with the standard curve prepared for the pure vitamin in the appropriate concentration in the region under the same conditions. Since the VB₂ solution is sensitive to visible light, the release experiments were performed using glassware with completely blackened surfaces so that the exposure of vitamin solutions to the visible light was minimized. The cumulative release rate of VB₂ (%) was calculated as follows:
M_t: the amount of VB₂ released from the carriers at time t; M₀: the amount of VB₂ initially loaded onto the carriers.

Results and discussion

Synthesis and characterization of β-CD polymers

The preparation of water-soluble CAβCDP was designed as a successive one step process and the reaction mechanism is illustrated in Scheme 1. These cationic polymers were obtained using the polymerization of β-CD and GN with EP in an alkaline medium, which molar ratio of EP/β-CD = 15 and ratio of GN/β-CD were chosen as 5, 10 and 15. At first, the β-CD was stirred with an excess amount of NaOH solution in order to form alcoholate sites. Then, GN was added to the obtained suspension. After addition of EP in this suspension, hydroxyl groups of β-CD or amino groups of GN react with one reactive group of the bifunctional agent and produce β-CD/epoxide or GN/epoxide compounds. The obtained side chain can further react in different ways: (1) the epoxide ring of β-CD/epoxide can react with another hydroxyl group of a second β-CD that resulting in a glyceryl bridge connecting two β-CD cavities (2) the epoxide ring of GN/epoxide can react with another amino group of a second GN molecules and connected two GN compounds (3) the epoxide ring of GN/epoxide can react with another hydroxyl group of a second β-CD molecules and β-CD/epoxide can react with another amino group of a second GN molecules to produce β-CD and GN in same compounds. In the last case, the modified and unmodified β-CD and GN molecules were crosslinked into polymer using remained EP. So, GN was grafted onto the β-CD polymer to achieve cationic β-CD polymer. The general trend was that the amount of GN fixed to the CAβCDPs increased with increase of the initially added the GN.

Scheme 1 Schematic illustration of preparation of cationic β-CD polymer through crosslinking of β-CD and GN with EP.

FT-IR analysis. The FT-IR spectra peak position for NEβCDP and CAβCDP-5, 10 and 15 are shown in Table 2. All of them are chiefly characterized with dominant O–H stretching vibration (3500–3300 cm⁻¹), C–H stretching vibration (2925–2855 cm⁻¹) and C–O stretching vibration (1157–1030 cm⁻¹), because both β-CD and glycerol units are formed by functional units possess a number of hydroxyl groups. On the other hand, there are four characteristic peaks for GN compounds as follows:⁷⁹ ν_NH at about 3300 cm⁻¹, ν_C=N at 1689–1650 cm⁻¹, δ_NH at about 1640 cm⁻¹ and ν_C–N at about 1300 cm⁻¹. The FT-IR spectra of CAβCDPs have characteristic groups of β-CD monomers and cationic GN groups. In the CAβCDPs a new vibration peak at 1660 cm⁻¹ is indicative of imine group at GN, which confirmed insertion of GN into polymers due to reaction between GN and EP. These results establish the produce of cationic β-CD polymer by grafted GN onto β-CD polymers as a bridging.

Table 2 FT-IR spectra peaks position for NEβCDP, CAβCDP-5, 10 and 15

Peaks (cm^−1)	NEβCDP	CAβCDP-5	CAβCDP-10	CAβCDP-15
3357	O–H stretch	O–H and N–H stretch	O–H and N–H stretch	O–H and N–H stretch
2925–2855	CH2 stretch (asym and sym)	CH2 stretch (asym and sym)	CH2 stretch (asym and sym)	CH2 stretch (asym and sym)
1674	C–C stretch	C–C stretch	C–C stretch	C–C stretch
1660	—	N–H bend	N–H bend	N–H bend
1157–1030	C–O stretch	C–O stretch	C–O stretch	C–O stretch

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NMR spectroscopy

¹³C NMR analysis. The chemical structures of the CAβCDPs were confirmed by ¹³C NMR spectroscopy. The ¹³C NMR spectra of β-CD derivatives show an asymmetric cavity as indicated by the signal separation of the carbon atoms characteristic. The ¹³C NMR spectra of NEβCDP, CAβCDP-5, 10 and 15 were obtained and the results are shown in Fig. 2. In the NEβCDP, the separated peaks at 55–81 ppm are characteristic of the signal for C-2, C-3, C-4, C-5 and C-6 of β-CD cavities, and also carbons of the glyceryl bridge that connecting two β-CD cavities. The peak near 101.7 ppm is related to the C-1 which is connected to the β-CD vertical glycosidic linkage. These results are in a good agreement with the synthetic composition of β-CD and EP, and the previously reported NMR results for this β-CD polymer.⁷⁷ In the spectra of CAβCDPs there are the similar separation signals in comparison to NEβCDP, except, the distinct signals at 158.5 ppm in CAβCDPs are assigned to the carbons of GN groups and the signals at 44.6 ppm related to C–N are assigned to the linkage between GN and EP. These results demonstrated that cationic β-CD polymers containing GN groups have been successfully prepared.

Fig. 2 ¹³C NMR spectra of NEβCDP, CAβCDP-5, 10 and 15 in DMSO-d₆.

¹H NMR analysis. ¹H NMR spectra were also used to characterize the structure of cationic β-CD polymer. The ¹H NMR spectrum of NEβCDP (Fig. 3) shows the peaks around 5.7 ppm assigned to the O–H proton in C-2 and 3 (OH-2 and 3), peak at 4.8 ppm assigned to the C-1 proton (H-1) and peak at 4.5 ppm is related to the proton of O–H in C-6 (OH-6) of the glucose unit and the broadened peaks between 3 and 4 ppm corresponding to protons from C-2, 3, 4, 5 and 6 of the pyranose rings of β-CD (H-2–6) and protons of glyceryl bridge. In the CAβCDPs, addition to the above-mentioned peaks, the weak, broadening and even level to the base line peaks in the range of 7.1–8.5 ppm are appeared which assigned to the N–H protons of GN. These results are indicative that insertion of GN group in polymer has been succeeded. The broadening and splitting peaks are resulted from the relaxation of N¹⁴ and the slow exchange rate of the proton on C=NH₂⁺.⁷⁹ However, the intensity of these peaks in CAβCDPs increases along with increasing of GN content which this outcome is consistent with Kjeldahl data (Table 1). These results were confirmed the reaction of β-CD and GN with EP to produce CAβCDPs.

Fig. 3 ¹H NMR spectra of NEβCDP, CAβCDP-5, 10 and 15 in DMSO-d₆.

TGA and DTG analyses. The TGA and DTG analyses of NEβCDP, CAβCDP-5 and CAβCDP-15 were performed and results have been shown in Fig. 4A and B. It can be seen from Fig. 4A, the NEβCDP illustrated two main steps of weight loss in TGA curve. The weight loss of NEβCDP before 240 °C during the first step was mainly due to water evaporation which may be formed the free water of NEβCDP, and the second mass loss of about 55.1% above 240 °C was ascribed to the thermal decomposition of NEβCDP. After graft of GN onto β-CD polymer, the achieved CAβCDP-5 and 15 also show two main stages of mass losses. The first mass loss was found from 50 to 230 °C which is due to the water dehydration of such hydrophilic polymers. The second mass loss was observed at temperature 230–500 °C, predominantly attributed to the decomposition of CAβCDP. Moreover, the DTG curve of NEβCDP exhibits two notable peaks centered at 271 and 342 °C. These two thermal events were related to weight losses from the polymer decomposition were as follows; (i) decomposition of EP polymer unit at 271 °C, and (ii) mass losses due to β-CD decomposition at 342 °C. After insertion of GN into β-CD polymer, one main steps of weight loss was observed in DTG curves of CAβCDPs. It was found that all the CAβCDPs have lower peak degradation temperature in compare with the NEβCDP. The degradation temperature of CAβCDP decreases along with increase of GN content in the polymer. The DTG curve of CAβCDP-15 shows the narrower peak area in comparison with the CAβCDP-5. On the other hand, it is notable that the CAβCDPs have high thermal stability when temperature is above 310 °C, where the CAβCDP-5 and 15 retain almost 58.1% and 63.3% of their initial masses until 500 °C, while pure NEβCDP retains 55.1% of its initial mass at the same temperatures stated above. These results confirm the preparation of CAβCDP from reaction between β-CD, GN and EP.

Fig. 4 TGA (A), DTG (B), DSC (C) and DDSC (D) curves of NEβCDP, CAβCDP-5 and 15.

DSC and DDSC analyses. DSC and DDSC curves of pure NEβCDP, CAβCDP-5 and 15 are presented in the Fig. 4C and D. These compounds show an endothermic event occurring at about 90 °C due to the vaporization of the residual water.⁸⁰ DSC thermogram of NEβCDP (Fig. 4C) revealed an endothermic peak at about 298 °C that confirmed the results of TGA. In the DSC curves of CAβCDP-5 and 15, the exothermic peak of the polymer decomposition emerged in lower temperature in comparison with NEβCDP. As shown in DDSC curve of CAβCDP-15 (Fig. 4D), the peak area of the decomposition polymer exhibited narrower peak area than the CAβCDP-5. This phenomenon should be due to the preparation of CAβCDP from reaction between the GN and EP.

Water solubility studies. The solubility data of β-CD, NEβCDP, CAβCDP-5, 10, 15 are summarized in Table 4. The β-CD solubility was significantly increased from 17.7 mg mL⁻¹ to 875 mg mL⁻¹ by polymerization of β-CD in the presence of EP. On the other hand, the solubility of β-CD polymer was increased with insertion of GN in structure of polymers. It was noted that the CAβCDPs solubility were higher than NEβCDP. In addition, it is clearly seen that CAβCDP water solubilities are increased along with increasing of GN content (Table 4).

Preparation and characterization of complexes

We have prepared inclusion complexes between VB₂ and CAβCDPs using a co-precipitation method, and then some properties of these complexes were investigated.

FT-IR analysis. FT-IR spectra were achieved to characterize interactions between VB₂ and polymers in the solid state. Fig. 5 depicts FT-IR spectra of VB₂, CAβCDP-5, 10 and 15/VB₂ and also CAβCDP-15 (for better comparison). As well known, it is not possible to confirm the presence of an inclusion compound in β-CD and β-CD derivatives by FT-IR,³³ but clear differences between the spectra for CAβCDPs/VB₂ and the free VB₂ were observed. In the CAβCDPs/VB₂ spectra absorption peaks of VB₂ were disappeared, which may be due to the vitamin was captured and distributed in the networks and cavities of the β-CD polymers.

Fig. 5 FT-IR spectra of CAβCDP-15 (for better comparison), VB₂, CAβCDP-5/VB₂, CAβCDP-10/VB₂ and CAβCDP-15/VB₂.

NMR spectroscopy. To confirm the presence of vitamin complex, the ¹H NMR and ¹³C NMR spectra of VB₂ and its complexes are shown in Fig. 6 and 7. As it can be seen from Fig. 6 and 7, the assignments of ¹H and ¹³C-chemical shift of VB₂ agree with previous reports in the literature.^74,81 The ¹H NMR spectrum of VB₂ in deuterated DMSO consists of the protons H-6 and H-9 of benzene ring can be readily assigned to peaks at 7.85 and 7.89 ppm, the aliphatic protons on carbons 1 to 5 were appeared at 4.91–4.22 ppm and also methyl group protons (H-13 and H-12) were revealed at 2.46 and 2.38 ppm.

Fig. 6 ¹H NMR spectra of VB₂, CAβCDP-5/VB₂ and CAβCDP-15/VB₂.

Fig. 7 ¹³C NMR spectra of VB₂, CAβCDP-5/VB₂ and CAβCDP-15/VB₂.

As shown in Fig. 6, the ¹H NMR spectra of VB₂ in the presence of CAβCDP-5 and 15 were examined in order to propose the structure of the complexes. ¹H NMR spectra of the complexes showed the proton peaks both of the CAβCDPs and VB₂, which several ¹H chemical shifts of VB₂ have been changed. It confirmed that the complexes were formed. Moreover, in the presence of CAβCDPs the proton peaks of H-1–5 were overlapped with the proton peaks of CAβCDPs, but the proton peaks of H-6, H-9, H-12 and H-13 were clearly visible in the spectra. Therefore, only these protons were examined upon addition of polymers and the results have been shown in Table 3. As it follows from Table 3, the chemical shift changes of H-6 and H-9 on VB₂ have been found 0.08 ppm and 0.02 ppm in the presence of CAβCDP-5 and also 0.1 ppm and 0.04 ppm in the presence of CAβCDP-15, respectively. The downfield chemical shifts of these protons after complexation of VB₂ represent the environment around these protons which are changed by the guest molecules included. These results indicate that the benzene ring of VB₂ molecule is located inside the macrocyclic cavity through inclusion complex formation. However, previous researches have shown that the benzene ring of VB₂ inserted into the cavity of β-CD, whereas its bulky ribityl side chain and the remaining two aromatic rings are placed outside the cavity.^74,81,82

Table 3 ¹H NMR and ¹³C NMR chemical shift (ppm) of VB₂, CAβCDP-5/VB₂ and CAβCDP-15/VB₂

	H-9	H-6	C-16	C-15	C-9	C-6	C-12
VB2	7.89 ppm	7.85 ppm	159.96 ppm	159.86 ppm	130.67 ppm	117.46 ppm	18.79 ppm
CAβCDP-5/VB2	7.97 ppm	7.87 ppm	160.33 ppm	160.21 ppm	130.82 ppm	117.77 ppm	18.97 ppm
CAβCDP-15/VB2	7.99 ppm	7.89 ppm	160.25 ppm	160.19 ppm	130.81 ppm	117.78 ppm	18.93 ppm

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The ¹³C NMR spectrum of VB₂ in deuterated DMSO have been shown the signals of carbons 1–16 (Fig. 7). So, Fig. 7 depicts ¹³C NMR spectra of CAβCDPs/VB₂ complexes, wherein both CAβCDPs peaks and VB₂ peaks were observed which reveals a possible displacement of VB₂ carbons resulting from vitamin inclusion and interaction.

As can be seen in Table 3, the chemical shift of VB₂ carbons have changes in the presence of CAβCDPs compared to the free vitamin. In the presence of CAβCDPs the downfield shifts of VB₂ carbons (C-6, C-9, C-12, C-15 and C-16) were due to interactions between the species through changes in the electronic density of VB₂ upon complexation. Finally, it can be deduced from the ¹H NMR spectra and previous studies that the benzene ring entered into the inner cavity of β-CD. On the other hands, the shielding of carbons were attributed to the inclusion and non-inclusion complexation. Thus, whole of VB₂ was included within the β-CD cavity and polymer networks.

DSC analysis. DSC analysis is used to confirm the host–guest interaction and thermal stability as well as to check the crystallinity of VB₂ in the polymer matrix. The DSC thermograms for CAβCDP-15 and CAβCDP-15/VB₂ are illustrated in Fig. 8. It is well documented that the DSC curve of VB₂ shows two transitions,⁷⁴ the first one exothermic at 256 °C which is due to crystallization and the second one endothermic at 279 °C which is due to the VB₂ melting. In the case of encapsulated VB₂, the peaks correspond to VB₂ transitions have been disappeared and the peaks in the curve are consistent with transitions of CAβCDP. It is indicated that the preparation of VB₂ loaded in the polymer and also the amorphous dispersion of VB₂ in the CAβCDP.

Fig. 8 The DSC curves of CAβCDP-15 (for better comparison) and CAβCDP-15/VB₂.

DLS analysis. This is one of the most popular technique which is used to determine the size distribution profile of small particles in suspension or polymers in solution. Fig. 9 shows the results that have been measured by DLS for an aqueous solution of the VB₂ and also CAβCDP-5 and CAβCDP-15 containing VB₂. These solutions were optically clear at ambient temperature; but, the DLS intensity distribution indicated the existence of small particles with a hydrodynamic radius of approximately 1281 nm, 255 nm and 338 nm for VB₂, CAβCDP-5/VB₂ and CAβCDP-15/VB₂, respectively. The size of loaded-VB₂ in the polymers were dramatically lower than VB₂ alone. Based on the evident of size determination studies, the size of loaded-VB₂ in the polymer was increased with increasing the GN ratio in the cationic polymers from 5 to 15. These results suggest that the VB₂ can be formed as an efficiency complex with the CAβCDP without any aggregate.

Fig. 9 Size distributions of VB₂, CAβCDP-5/VB₂ and CAβCDP-15/VB₂ complexes in terms of volume in aqueous solution at 25 °C.

Zeta potential. Zeta potential is an essential instrument to investigate the surface charges and host–guest interaction types. Fig. 10 shows the zeta potential values that obtained for the pure VB₂ and loaded-VB₂ in the CAβCDP-5 and CAβCDP-15. These measurements were conducted in DI water to avoid the influence of pH transitions. The zeta potential of VB₂ alone is around −30.6 mV, which could be increased to +10.9 and +11.3 mV after being complexed with CAβCDP-5 and 15, respectively. It was noted that the native VB₂ has a negative zeta potential, while the loaded-VB₂ in CAβCDPs has a positive zeta potential value which is indicated that the complexes surface were positively charged due to the availability of the C=NH₂⁺ groups in the β-CD polymer. These results suggested that the VB₂ are included in the cavities and networks of CAβCDP, as well as the electrostatic attraction between the anionic groups of VB₂ and cationic groups of CAβCDP. As it is expected, the zeta potential values of complexes increased with increasing the GN amount due to the increased amount of cationic groups in the CAβCDP.

Fig. 10 Zeta potential of VB₂, CAβCDP-5/VB₂ and CAβCDP-15/VB₂ in aqueous solution at 25 °C.

Water solubility study. The water solubility data for VB₂ and VB₂ in the presence of native β-CDs, NEβCDP, CAβCDP-5, 10 and 15 are summarized in Table 4. The VB₂ is a poorly soluble vitamin and its solubility in water obtained 0.078 mg mL⁻¹ which is in agreement with the literature.^83,84 From Table 4, it can be understand that CAβCDP/VB₂ solubility is much higher than the VB₂ and β-CD/VB₂ solubility and slightly higher than the NEβCDP/VB₂ solubility. As a result, it can be concluded that the VB₂ solubility increases because of the formation of inclusion complexes between VB₂ and CAβCDPs. As it is concluded from NMR data, benzene ring of VB₂ molecule penetrates into the β-CD cavity. This should be favorable for enhancement of the aqueous solubility of vitamin. In addition, decreased crystallinity coupled with zeta potential change are important factors which is playing a positive role in increasing VB₂ solubility. It is clearly seen that the water solubility of all complexes were obviously increased along with increasing of GN amount (Table 4).

Table 4 Solubility data of β-CD, NEβCDP, CAβCDP, VB₂ and VB₂ in the presence of native β-CDs and β-CD polymers

Samples	Solubility (g/1000 mL, 30 °C)
VB2	0.078 ± 1.1
β-CD	17.7 ± 1.3
NEβCDP	875 ± 3.6
CAβCDP-5	925 ± 2.6
CAβCDP-10	1061 ± 3.4
CAβCDP-15	1285 ± 1.8
NEβCDP/VB2	394 ± 3.1
CAβCDP-5/VB2	484 ± 3.7
CAβCDP-10/VB2	536 ± 4.1
CAβCDP-15/VB2	673 ± 2.9

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In vitro release studies

In this study, cumulative release of VB₂ from CAβCDP-5 and 15 in gastric acidic (hydrochloric acid buffer, pH = 1.2), in intestinal (phosphate buffer, pH = 7.4) and in alkaline conditions (carbonate buffer, pH = 10) were performed. For comparative purposes in these experiments the cationic polymers with different amount of GN were chosen. The release of VB₂ from polymers were characterized over 26 h at 37 °C, and the percentage of vitamin release from these samples against the time have been shown in Fig. 11A and B.

Fig. 11 Cumulative release of VB₂ from (A) CAβCDP-5 and (B) CAβCDP-15 for 1600 minute.

As it is expected, for two cationic polymers which possess the cationic networks and hydrophobic cavity of β-CD, typically, a biphasic release profile can be observed in which a burst release stage is followed by a sustained release phase over the time (Fig. 11). The burst phase should be due to the release of free VB₂ molecule and the incorporated VB₂ into the networks through non-inclusion interactions. For the VB₂ molecule that are associated with β-CD units through the inclusion interactions, the release occurs through a slower dissociation and diffusion. In the case of the CAβCDP-5 (Fig. 11A), more than 98.53% and 97.63% of the vitamin was diffused out of the system at pH 10 and 1.2 after 23 h respectively. While approximately 81.5% of loaded-VB₂ was released from this polymer after the same time at pH 7.4. On the other hand, as it can be seen, the vitamin release rate from CAβCDP-15 is slower than CAβCDP-5 with similar release profiles in the case of all the medium that have been studied (Fig. 11B). Within 23 h nearly 89.1, 71.5% and 91.7% of loaded-VB₂ was released from CAβCDP-15 at pH 1.2, 7.4 and 10 into the medium respectively. It can be observed that the amount of released VB₂ from the cationic polymers at pH 10 and 1.2 buffer solution are higher than the pH 7.4 buffer solution, and also at buffer solution with pH 10 the release is higher than buffer solution with pH 1.2. Therefore, released VB₂ from cationic polymers is dependent on the medium pH and GN content which the GN acts as a pH-response site to control vitamin release. The decelerated release of VB₂ under three conditions from CAβCDP-15 in comparison with CAβCDP-5 may be due to the stronger electrostatic interactions between vitamin and cationic polymer which is consistent with the result of NMR analysis.

Conclusions

In this work, we have prepared inclusion complexes between VB₂ and CAβCDPs using a co-precipitation method. The obtained results from solid-state and solution show that the vitamin capture and distribution in the cavities and networks of cationic polymer. The size of loaded-VB₂ in cationic polymers are dramatically lower than native VB₂ alone. The obtained complexes have a positive zeta potential, while values of zeta potential for VB₂ is negative, which is indicated that the complexes surface is positively charged. In addition, due to formation of complexes between VB₂ and highly water soluble cationic polymer, the vitamin solubility is improved dramatically. It is clear that increasing GN content in cationic polymers increases the water solubility of all complexes. Proposed water-soluble CAβCDP is synthesized by a simple and friendly environmental method. The typical analyses revealed insertion of cationic GN in the achieved polymeric structure. In here, the cumulative release of VB₂ from CAβCDPs in gastric acidic, intestinal and alkaline conditions were performed. The results have been illustrated a burst release stage and followed by a sustained release stage over the time. Therefore, we think this new prepared encapsulated VB₂ can be considered with various potential applications including in both food industry and in pharmaceutical field.

Acknowledgements

The authors express appreciation to the Physiology Research Center, Deputy of Research and Technology, Kerman University of Medical Sciences and Shahid Bahonar University of Kerman, to financial support of this study.

Notes and references

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