1. Introduction
Hydrogels are tridimensional networks formed by interconnected polymer chains to form an insoluble network. They absorb large amounts of water while still maintaining the 3D structure and the original shape while changing their volume [
1]. There are several possibilities to form a polymeric network, both by chemical bond or physical interaction. Chemical hydrogels have the capability to be chemically and physically stable unless specific chemical bonds are added, or are naturally present, to allow a full or partial network degradation upon established stimuli [
2]. For biomedical applications, among the most used polymers for hydrogel preparation, the great family of polysaccharides should be mentioned. Hyaluronic acid [
3], chitosan and derivatives [
4], dextran [
5], alginate [
6], cellulose [
7] and heparin [
8] are among the most used. Polysaccharides are also employed for colon drug delivery because most of them are specifically degraded from intestinal microflora [
9,
10,
11]. From a pharmaceutical point of view, the polysaccharides can be considered as a gold standard in the delivery of water soluble molecules including macromolecules such as proteins and other biomolecules such as peptides or DNA/RNA fragments [
12]. These kinds of molecules are mostly excluded from being delivered by other drug delivery systems (DDSs) such as those with hydrophobic groups providing a good environment for non-water-soluble molecules. Polymeric micelles are a great tool for the technologist, since they allow the drug delivery of highly hydrophobic drugs even by intravenous administration [
13]. Other commonly used DDSs are undoubtedly liposomes and solid dispersions [
14,
15].
Polymeric micelles are self-assembling systems made from amphiphilic polymers that can carry, within their hydrophobic core, water insoluble molecules. In literature there are lots of examples that describe polymers self-assembling in micelle systems [
16]. The delivery of hydrophilic drugs by micelle systems is somewhat troubling. In particular, when dual drug release is needed, for a combination therapy, choosing the right DDS would be problematic. This is the issue we will address in this paper.
The aim of this paper is to propose a DDS that can simultaneously load both hydrophilic and hydrophobic drugs. It would be accomplished by linking together the advantages of hydrogels and polymeric micelles. In theory, it could be simply accomplished by hydration of a preformed hydrogel with a micelle suspension but, in this way, the leakage of the polymeric micelles, e.g., during storage or even after administration, is predictable. Thus, we thought to provide the hydrophilic shell of a polymeric micelle with photocrosslinkable moieties to form a “nanogrid” in which the micelles represent the hydrophobic netpoints between the hydrophilic hydrogel.
The chosen polymer was inulin (INU), since it is biocompatible, nontoxic, FDA-approved for human use and, overall (for our aim), it is specifically degraded by gut microflora (
bifidobacter degradation). INU is a hydrophilic polyfructose obtained from renewable sources widely used in the alimentary industry for its prebiotic activity (stimulating the growth of beneficial bacteria). We previously synthesized an amphiphilic derivative of INU with vitamin E (VITE) which demonstrated high self-assembling properties plus a series of features such as biocompatibility both in vitro and in vivo that makes this derivative an ideal candidate for the delivery of highly hydrophobic drugs [
17]. On the other hand, INU was also proposed for the formation of pharmaceutical hydrogels that showed favorable characteristics for colon drug delivery.
Here we show how the methacrylated INVITE, named INVITEMA, can form a micellar-hydrogel nanogrid specifically designed for the delivery of hydrophilic and hydrophobic drugs into the colon.
2. Materials and Methods
2.1. Materials
Triethylamine (TEA), dicyclohexylcarbodiimide (DCC), N,N-dimethyl-formamide (DMF), d-α-tocopherol succinate, methacrylic anhydride (MA), pyrene, diethyl ether, dimethyl sulfoxide (DMSO) and dimethylsulfoxide-d6 (DMSO-d6), Methanol HPLC grade, beclomethasone dipropionate (BDP) and inulin from Dahlia tuber (INU, Mw ~5000 Da) were from Sigma-Aldrich (Milan, Italy) and used without further purification. N-hydroxysulfosuccinimide sodium salt and curcumin (CUR) were from TCI (Zentek S.R.L, Milan, Italy). Dialysis membrane MWCO 3500 Da were from Spectrum Labs (VWR international, Milan, Italy). The used water was from a Milli-Q gradient A-10 system (18.2 MΩ cm, organic carbon content ≤4 μg/L) (Merck-Millipore, Milan, Italy).
2.2. Apparatus
Fourier Transform Infrared Spectroscopy (FTIR) spectra were registered as KBr pellets with a Perkin-Elmer 1600 IR Fourier Transform Spectrophotometer (Perkin-Elmer, Monza, Italy). The resolution was 1 cm−1. Recording range 4000–400 cm−1.
Centrifugations were performed with a Beckman Avanti 30 (Beckman, Milano, Italy) equipped with a temperature control.
UV-Vis spectra were obtained by a Perkin–Elmer Spectrometer Lambda 25 (Perkin-Elmer, Monza, Italy).
1H-NMR were acquired with a Varian Mercury 300 MHz instrument (Varian, Segrate, Italy).
Hydrodynamic size and polydispersity index (PDI) of INVITEMA micelles were evaluated by a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK).
UV irradiation of INVITEMA nanomicelles was accomplished with a “Polymer” reactor (Helios-Italquartz, Milan, Italy) provided with an UV lamp of 125 W having an emission range 250–364 nm. Emission peak 310 nm.
DSC studies were from a DSC 822E Mettler-Toledo instrument (Milano, Italy) provided with a refrigerated cooling accessory.
TEM studies were performed by a Jeol-Jem-1200EXII (Tokio, Japan). In particular, a copper grid was used as support to deposit a drop (1 mg/mL) of INVITEMA suspension, after drying at 25 °C in a desiccator the grid with the deposited sample was negatively stained with uranyl acetate and analyzed.
SEM pictures were acquired by sticking the samples on a stub provided with a carbon adhesive disc and subsequently subjected to coating by a 2 nm layer of palladium by using an electron beam evaporator. The used instrument was a Sigma Zeiss (Zeiss, Milan, Italy) field emission scanning electron microscope (FE-SEM). Acceleration voltage of the SEM probing e-beam was set at 3 kV; an in-lens detector and 30 μm slit aperture were used.
HPLC studies were performed with a LC Agilent (Agilent, Milan, Italy) equipped with a quaternary pump VL1260, a 1200 series Diode Array Detector, a 1220 Series standard autosampler SL, an ELSD detector 1260 Infinity, a Lab Advisor Basic Software and using an OpenLAB CDS LC ChemStation (Agilent, Milan, Italy) on Workstation HP Compaq 6300.
2.3. Synthesis of INVITEMA Conjugates
The INU-α-tocopherol succinate conjugate (INVITE) was synthesized as previously reported [
17]. For the synthesis of the new conjugates bearing methacrylic-UV-reactive groups on the side chain (noted as INVITEMA1; INVITEMA2 and INVITEMA3), 200 mg of INVITE were solubilized in 10 mL of anhydrous DMF and then, calculated amounts of trimethylamine (TEA) and methacrylic anhydride (MA) were added according to the following ratio being “RU” Repeating Unit:
R1 = Moles of MA/Moles of RU INVITE = 0.2; 0.35; 0.5
R2 = Moles of TEA/Moles of RU INVITE = 0.5
The reaction mixture was maintained at 25 °C for 20 h under constant stirring and under N2 atmosphere. Then, the product was recovered by precipitation in Et2O and purified by 5 washing cycles using the same solvent. After each washing, the polymer powder was recovered by centrifugation at 4 °C, 5 min, 4000 rpm. The products were obtained with an 86–98% w/w yield range and were characterized by 1H-NMR spectroscopy and FTIR, critical aggregation concentration (CAC) determination, TEM and SEM, dimensional analysis, and stability studies. Derivatization degree (DD mol/mol %) was calculated from 1H-NMR as ratio between the integral of the peaks at 5.70 and 6.08 ppm (MA, s, 2H) and the integral of the peaks at 3.5–4.0 ppm (fructose ring, m, 7H).
2.4. CAC Determination
CAC determination for INVITEMA conjugates was accomplished by using the pyrene method as already reported [
18].
In particular, excitation was set at 334 nm and emission was registered in the wavelength range 350−450 nm. Both excitation and emission slit was fixed at 5 nm. Pyrene was added to the INVITEMA sample solutions: final concentration of pyrene 2 × 10−6 M. 83.4 μL of pyrene stock solution, in acetone, (6 × 10−3 M) was combined with water, to gain an actual pyrene concentration of 2 × 10−5 M. This solution was maintained under vigorous stirring and, to remove the acetone, it was maintained under continuous nitrogen flow. Different INVITEMA water solutions in the concentrations range 10−7−2.5 mg/mL were prepared and to 0.9 mL of these suspensions 100 μL pyrene water solution were added. This mixture was left equilibrating overnight. Finally, the ratio of the intensities I1 and I3 against INVITEMA concentration was plotted.
2.5. Preparation and Characterization of Empty or Drug-Loaded INVITEMA Micelles by Direct Dialysis Method
Micelles from INVITEMA3 conjugate were prepared with concentration above its critical aggregation concentration (1.2 × 10−2 mM). To obtain polymeric micelles, a DMSO solution of INVITEMA3 conjugate was prepared in the presence or not of 10% w/w of beclomethasone dipropionate (BDP) and dialyzed vs. distilled water for 3 days. In particular, the polymer-drug or polymer DMSO solution, was poured into a 3500 Da MWCO regenerated cellulose (RC) dialysis membrane sealed by clamping the two extremities. The filled dialysis tube was put in a 3 L vessel and left under mild stirring performing regular water changes. After three days, the micellar water dispersion was filtered by a 0.45 µm RC membrane to eliminate non-loaded drug and, subsequently, lyophilized and recovered with a 90% w/w yield.
2.6. Differential Scanning Calorimetry Studies
Five mg of each sample were exactly weighed. The tested temperature range was 25–250 °C for BDP and INVITEMA products setting the heating rate at 5 °C/min. For Tg determination, a cycle method was applied as specified: (1) heating to 160 °C (30 °C/min); (2) cooling to −20 °C (30 °C/min) (3) heating to 160 °C with (30 °C/min). Sample cells were purged with nitrogen (flow 50 mL/min). The midpoint of the deflection in the heat flow vs. temperature curve, as belonging to the second heating cycle, was considered as the Tg. Non-hermetic aluminum pans were used, each measurement was repeated three times.
2.7. Size Distribution and Morphology
INVITEMA aqueous dispersions, at concentration of 1 mg/mL were allowed to equilibrate at 25 °C, overnight, under slow stirring. After this time, the micellar dispersions were filtered by a 0.45 μm filter and analyzed by the indicated instrument. The measurements were performed in triplicate for each sample.
2.8. Preparation of INVITEMA Nanogrids
INVITEMA (60 mg/mL) samples were dispersed in degassed Milli-Q
® grade water and poured in Pyrex vials. The Vials containing the micelle suspension were irradiated for 30 min, 1 h or 2 h, without any photo-initiator, see
Section 2.2 for the used equipment. After the irradiation time, the obtained nanogrid was recovered and freeze-dried.
2.9. Water Uptake Studies
Water uptake studies were performed in water at 37 °C for 24 h. In particular, the established amount of INVITEMA nanogrids (≈30 mg) were put in a glass tube open, the used tubes are opened in the lower part and provided with a support glass filter of 10 mm in diameter and a G2 porosity. The tubes were so put in water at 37 ± 0.1 °C and the nanogrids left to swell. After each established time point, namely 1, 2, 3 and 24 h, the tubes were allowed to percolate the excess water at atmospheric pressure, then, the tubes were placed inside a centrifuge tube and centrifuged for 5 min at 3000 rpm.
Water uptake was calculated as: q = Ws/Wd, where Ws is the weight of the swelled nanogrids while Wd is the weight of the dry nanogrids.
The reported values are the mean of three measurements for each time point and are in accordance with a standard error of 5%.
2.10. Drug Loading Evaluation on INVITEMA3 Nanogrid
For the drug loading evaluation of INVITEMA3 samples, 10 mg of freeze-dried micelles were dispersed in 10 mL of dimethyl sulfoxide [
19]. Drug loaded BDP was calculated by HPLC as specified in
Section 2.12. The loaded amount of BDP was calculated by a calibration curve of BDP as specified in
Section 2.12. We also attempted to load curcumin in the same way than BDP but, not surprisingly, it degraded upon UV irradiation. Each measurement has been performed in triplicate.
The drug loading (DL w/w %) in INVITEMA3 was calculated based on the following formula:
DL (w/w %) = (BDP HPLC-calculated loaded weight/weight of nanogrids plus BDP) × 100
The Encapsulation efficiency (EE%) was calculated as:
EE% = (BDP HPLC-calculated loaded weight/theoretical BDP amount) × 100
2.11. Evaluation of Drug Stability upon UV Irradiation
To evaluate a possible UV degradation of BDP during UV-induced crosslinking of BDP loaded INVITEMA nanomicelles, a solution of BDP in methanol or acetone at 10%
w/
v was prepared. This solution was irradiated in the same conditions of the INVITEMA bioconjugates (see
Section 2.8), then, the solution was dried under vacuum, the drug recovered and analyzed as described in
Section 2.12.
2.12. Drug Release Studies from INVITEMA Nanogrids
10 mg of BDP loaded INVITEMA nanogrids, were suspended in 1 mL of Milli-Q® grade water and the solution poured into a dialysis membrane with a molecular weight cut-off of 3500 Da. The dialysis tube was put in a vessel with 10 mL of PBS pH 7.4/Tween 80 0.5 w/w %, at 37 °C.
At scheduled time points, namely 1, 2, 4, 8, 24, 48, 72 and 120 h, the whole release medium was collected and replaced with fresh release media at 37 °C. The amount of released BDP has been calculated by HPLC as follow: column C18 (250 × 46 mm, 5 µm, Zorbax) from Agilent (Milan, Italy), flow rate 1 mL/min, wavelength 238 nm, mobile phase MeOH/H2O 70:30 v/v isocratic. The calibration curve of BDP was performed by dissolving it in HPLC grade MeOH in the concentration range 0.5–150 µg/mL, at least 6 concentration points were set inside the interval and each point was performed in triplicate (R2 = 0.9987).
To verify the maintenance of sink conditions along the experiment, BDP dissolution was evaluated as follow: based on the calculated DL %, 100 µg of BDP were suspended in 1 mL of Milli-Q® grade water and the solution poured into a dialysis membrane with a molecular weight cut-off of 3500 Da. The dialysis tube was put in a vessel with 10 mL of PBS pH 7.4/Tween 80 0.5 w/w %, at 37 °C. The experiment was carried on until the 100% drug dissolution was reached.
4. Discussion
We recently developed the INVITE platform based on the conjugation of inulin and vitamin E to obtain amphiphilic polymers able to self-assemble in polymeric micelles at low concentration, showing a high biocompatibility and a tailorable chemical functionalization [
23,
24,
25]. These systems are extremely versatile but, as with all the micelle systems, have a limitation of mostly including, within the hydrophobic core, hydrophobic molecules. While this fact is a great advantage for drugs showing low water solubility, it could constitute a problem when the delivery of a hydrophilic molecule is required, especially in combination therapy. Several examples can be found in the literature of combination therapy using micelle systems included in hydrogels [
26,
27,
28]. Only a few examples in the literature report on the formation of hydrogels from modified micelles for drug delivery [
29,
30,
31]. Undoubtedly, it is a great advantage to have a system formed from a hydrophilic matrix showing drug-accessible hydrophobic clusters that can allocate a wide variety of drugs.
This is what drove our work: we were looking for a drug delivery system to be proposed for colon delivery with the intention of delivering both hydrophilic and hydrophobic drugs in the course of, e.g., chronic diseases such as Crohn’s disease. In particular, we imagined a nanogrid system in which the main hydrophilic network is formed by the crosslinking of the external shell of inulin-based micelle while the hydrophobic portion is formed by the vitamin E core of the self-assembling system. Inulin is an optimal candidate for colon delivery because it is specifically degraded in the colon as result of bifidobacter action, INU is a prebiotic.
With this aim we functionalized INVITE micelles (INU functionalized with VITE) with methacrylic groups to form the nanogrids upon UV irradiation. This crosslinking technique was chosen since it does not require any purification step (no radical initiator was needed) and assures a good degree of sterilization. The covalent linking of the methacrylic moieties was confirmed by FTIR and
1H-NMR studies as discussed in
Section 3.2. The self-assembling properties of the INVITEMA system were assessed by CAC studies that returned a value of 1.4 × 10
−2 mM for INVITEMA3. This value is really low even when compared with commercially available surfactant, e.g., sodium dodecyl sulfate (SDS) shows a CMC of 7.96 mM, dodecyltrimethylammonium bromide (DTAB) shows a CAC of 13.88 mM while Tween−20 shows a CAC of 0.053 mM [
17]. The achieved result from CAC suggests a good stability of the micelle system upon dilution and in the time course of UV irradiation, this assumption is further corroborated by the DLS studies that demonstrated a dimensional stability of the micelles up to 30 days.
Nanogrid-hydrogel formation took place upon UV irradiation. The consumption of methacrylic double bonds was monitored by FTIR and, in particular by the disappearance of the characteristic bands at 816 and 1350 nm representative of the double bonds. Since the radical polymerization prompted by UV is dependent on both concentration and time of irradiation, the results obtained from the three derivatives showing a different degree of derivatization at different times are extremely variable. The polymers’ concentration was constant and equal to 60 mg/mL. What mostly influenced the crosslinking was the number of formed radicals during the irradiation. The concentration of 60 mg/mL, which corresponds to 6% w/v, was chosen to maintain a low viscosity of the suspension to optimize a UV ray deep penetration. As from FTIR, only the sample INVITEMA3 after irradiation for 2 h brought the complete consumption of the methacrylic double bonds. For this reason, this sample was chosen for the subsequent characterizations. In particular, TEM and SEM studies confirmed the formation of the nanogrids, shown as spherical protuberances immersed in the polymeric foils. Their distribution appears random but the maintenance of the spherical shape and the emergence from the foil surface lets us hypothesize that upon UV irradiation the micelles first form a “nanocapsule” due to the crosslinking of the micelles shell, and then the outer methacrylic groups will react with adjacent micelles forming the polymeric foil.
This assumption is also demonstrated by the SEM pictures in
Figure 6; after 30 min UV irradiation, spherical nanocapsules are mostly present, and upon increasing the time of irradiation the polymeric foil grows until a continuous layer is formed. The “nanocapsules theory” is also supported by the qualitatively evaluable size differences between the mean hydrodynamic size of the micelles before UV crosslinking that, as seen in DLS studies, results almost 10 nm and the size from the scale bar of SEM that is evaluable is almost 30 nm. This fact can be explained by considering that upon UV crosslinking the outer hydrophilic layer of the micelle can grow and retains its size after freeze drying, it being a swelled hydrogel. This fact is well explained by FTIR that shows the presence of unreacted double bonds after 30 min or 1 h UV irradiation. This assumption also justifies the increase of water uptake for the sample INVITEMA3/2 h. In fact, while the water uptake of the INVITEMA3/30 min and INVITEMA3/1 h is mostly affected by the swelling of the “nanocapsules”, sample INVITEMA3/2 h will expose to the environment a larger hydrophilic area due to the INU foils. It will lead to higher water uptake as shown in
Figure 7.
Drug release studies, as can be seen in
Figure 8, show a constant release rate of the hydrophobic drug BDP. After 120 h almost 60% of the loaded drug was released. It represents the characteristic trend of the micelle system plus the effect of partitioning of the hydrophobic drug along the hydrophilic layer composed by the INU portion. In the literature, similar drug delivery systems show similar release behaviors [
30,
31].
5. Conclusions
The aim of this paper was to prepare new hydrogels provided with hydrophobic netpoints to be used for the combined delivery of both hydrophilic and hydrophobic drugs. Starting from the chemical synthesis of a methacrylated derivative of the amphiphilic bioconjugate INVITE (formed from inulin and vitamin E), we produced “nanogrids” by UV irradiation of the resulting polymeric micelle dispersion.
The obtained methacrylated derivatives were physicochemically characterized. The gained INVITEMA bioconjugates exhibited a low critical aggregation concentration that suggests a high stability of the obtained micelles upon dilution. The methacrylated micelles have been crosslinked by UV irradiation and it has been found that, depending on the irradiation time, the whole double bonds can be consumed or not. Upon complete consumption, a nanogrid was formed showing foils of hydrophilic polymer embedding the micelles, as shown by SEM analyses. Drug release studies shown a constant release rate in the tested time. We are evaluating other drugs, such as curcumin, to test the post-irradiation loading since its loading before UV irradiation results in its degradation. Biological evaluations and degradation are under investigation.
These systems are proposed for colon drug delivery due to the biodegradability of INU by intestinal microflora. Undoubtedly, the possibility of loading hydrophilic drugs such as proteic drugs together with hydrophobic drugs such as BDP provides new options for the therapy of chronic diseases such as Crohn’s disease.