Fabrication, characterization, thermal stability and nanoassemblies of novel pullulan-aspirin conjugates

2.1 Materials

Aspirin (US Pharmacopoeia standards) used was gifted by Askari Pharmaceuticals, Pakistan. 1,1′-Carbonyldiimazole (CDI) was obtained from Aldrich while pullulan was purchased from IL, USA. Analytical grade N,N-dimethylacetamide (DMA), anhydrous lithium chloride and other chemicals were used as received from Fluka.

2.2 Instrumentation

The FTIR (KBr pellet technique) spectra (ν, cm⁻¹) were acquired on IR Prestige-21 (Shimadzu, Japan).

The ¹H NMR spectra (δ, ppm; DMSO-d₆, NS 16) of pullulan-aspirin conjugates were recorded on Bruker 400 MHz machine while for ¹³C NMR 5000 Scans were accumulated.

Thermal degradation studies were performed on a SDT Q 600 (TA Instruments, USA) thermal analyzer. The thermal spectra were recorded at the onset of significant weight loss under nitrogen at a constant heating rate of 10 °C/min from RT up to 800 °C.

The products were studied by transmission electron microscopy (TEM) for self-assemblies. TEM used was Philips 420 instrument with an acceleration voltage of 120 kV.

2.3 Dissolution of pullulan

Pullulan was dried under vacuum at 110 °C before dissolution for 1 h. Dry pullulan (2 g) was dissolved in DMA (30 mL) by stirring at 90 °C. Polymer was dissolved within 25 min with optical clarity.

2.4 Synthesis of pullulan-aspirin conjugates 1, a typical example

Aspirin (2.22 g, 12.33 mmol) was dissolved in DMF (40 mL) solvent and CDI (1.99 g, 12.33 mmol) was added in parts under nitrogen atmosphere. Reaction mixture was stirred for 24 h at RT for the synthesis of reactive imidazolide of aspirin. This reaction mixture was added slowly to the solution of pullulan (2 g, 12.33 mmol) in DMA. Reaction mixture was continuously stirred at 80 °C for 24 h under nitrogen. Isolation of the product was carried out by precipitation of reaction mixture into 200 mL ethanol. The precipitates of sample 1 were washed thoroughly with ethanol in order to remove any of the side products and un-reacted drug contents. Precipitates of pullulan-aspirin conjugate 1 were dried under vacuum at 50 °C. Similar reaction procedures were adopted for the rest of pullulan-aspirin conjugates. Yield: 1.95 g (76%); DS = 0.32 by ¹H NMR spectroscopy; FTIR (KBr): ν = 3431 (OH), 2926 (aromatic C–H), 1740 (C⚌O ester), 1462 (CH₂) cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆, δ): 1.98 (H-7), 3.35–5.70 (AGU-H-1-6), 6.94 (H-9,11), 7.50 (H-10), 7.86 (H-8) ppm; ¹³C NMR (DMSO-d₆, δ): 170.49 (C-8), 168.99 (C-7), 117.75 (C-1), 119.90 (C-3), 130.61 (C-5), 136.16 (C-6), 136.26 (C-4), 160.61 (C-2), 101.84 & 95.99 (C-10), 74.45-67.5 (C11–14), 60.56 (C-15), 21.08 (C-9) ppm.

2.5 Transmission electron microscopy

Nanoparticles of pullulan-aspirin conjugates 3 were prepared using dialysis process. Sample 3 (20 mg) was taken in a typical batch, dissolved in purified DMSO (5 mL). Dialysis process was continued for 4 days against water along with constant stirring. The nanoparticle suspension was concentrated. The TEM samples were prepared using drop casting on carbon coated copper grids. The TEM grids were dried under air before studying by TEM.

2.6 Thermal degradation kinetic analysis

The kinetics of thermal decomposition of pullulan-aspirin conjugates were examined by Friedman, Broido and Chang methods. Friedman kinetic model (Friedman, 1964) uses the following equation; $$\text{ln}\left(\frac{d\alpha }{\mathit{dt}}\right)=\text{ln}Z+n\text{ln}(1-\alpha )-\frac{E_{\text{a}}}{\mathit{RT}}$$ where, dα/dt is the rate of weight loss directly taken from the DTG curve; Z is the frequency factor of decomposition reaction; n is the reaction order; 1−α is the weight of sample left at a certain temperature that is also taken from the TG curve; E_a is the activation energy; R is the gas constant and T is the absolute temperature recorded.The order of the reaction was calculated from the Chang model as given below (Chang, 1994). $$\text{ln}\left[\frac{\frac{d\propto }{\mathit{dt}}}{{(1-\propto )}^n}\right]=\text{ln}Z-\frac{E_{\text{a}}}{\mathit{RT}}$$ A straight line was obtained when ln [(dα/dt)/(1−α)ⁿ] was plotted versus 1/T in case if the assumed n value is found correct. The slope and intercept of the straight line were used to calculate the E_a and Z values.

The Broido method (Broido, 1969) was also used to calculate kinetic parameters, i.e., E_a and Z as per following equation. $$\text{ln}\left(\text{ln}\frac{1}{y}\right)=-\frac{E_{\text{a}}}{\mathit{RT}}+\text{Constant}$$ where, y = (w_t−w_∞)/(w₀−w_∞), w_t = weight at a given time t, w₀ = initial weight and w_∞ is final weight.

3.1 Synthesis and characterization

The MPDs of aspirin with a hydrophilic polysaccharide, i.e., pullulan, were synthesized using one pot and homogeneous reaction conditions (Samples 1–4). Pre-dissolved pullulan was reacted with imidazolide of aspirin drug prepared after activation of its carboxylic acid functional group with 1,1′-carbonyldiimidazole (CDI). Reaction methodology is generalized in Scheme 1.

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Scheme 1 Synthesis of MPDs of aspirin with pullulan applying in situ activation of aspirin with CDI.

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Aspirin was first activated with a fascinating RCOOH activating reagent CDI. CDI reacts with RCOOH of aspirin to make its imidazolide. Imidazolide was then reacted with the –OH functional groups of the anhydroglucose units (AGU) of pullulan to generate ester linkage. In this way, ester conjugates as MPDs of aspirin were successfully synthesized. A base imidazole was generated in situ that neutralizes the unreacted drug therefore no extra base is needed during the reaction. Pullulan-aspirin conjugates 1–4 were fabricated by using different mole ratios of the reactants (AGU:CDI:aspirin). The results of esterification reactions are given in Table 1 for all newly fabricated MPDs of aspirin onto pullulan.

Aspirin has been previously attached with another cellulose derivative i.e., hydroxypropylmethylcellulose (HPMC). Although, it was pioneering work for the attachment of aspirin onto any ether derivative of cellulose the reaction resulted in low DS of aspirin onto HPMC (Hussain et al., 2011a,b). The DS of aspirin in HPMC-aspirin conjugates was obtained in the range of 0.04–0.14. However, considerably high drug loading was achieved in the present study. Ds. was found in the range of 0.32–0.40 for pullulan-aspirin conjugates with same mole ratios of drug to polymer. This increased drug loading can potentially reduce the size of dose.

Aspirin prodrugs synthesized by the CDI method were found soluble in different organic solvents, i.e., DMSO, DMF, acetone and DMA. The products were thoroughly characterized by using different spectroscopic techniques. Thermogravimetric (TG) analyses were used to determine the stability of the fabricated MPDs. Transmission election microscopy (TEM) was used to study nanoassemblies in solution. The detailed structure characterization is given below.

Aspirin conjugates 1–4 have shown ester peaks in FTIR spectra. The FTIR (KBr) spectrum of pullulan-aspirin conjugate 2 (Fig. 1) has shown ester absorptions at 1744, aromatic C–H absorption at 2926 and unreacted –OH absorption at 3437 cm⁻¹. Nevertheless, pullulan-aspirin conjugates 1–4 have displayed aromatic C–H absorptions, hydroxyl group absorption and CH₂ (polymer) absorption signals as well in the FTIR spectra that indicate the success of esterification reactions.

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Figure 1 FTIR (KBr) spectrum of pullulan-aspirin conjugate 2.

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The ¹H NMR (400 MHz) spectra of pullulan-aspirin conjugates were recorded in DMSO-d₆. The typical spectrum of sample 1 (Fig. 2) showed the presence of the aromatic ring coupled with pullulan polymer backbone as aromatic protons were detectable at δ 6.94–7.86 ppm. The aromatic H-9 and H-11 overlapped and absorbed at δ 6.94 ppm while H-8 and H-10 absorbed at 7.86 and 7.50 ppm, respectively.

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Figure 2 The ¹H NMR (400 MHz) spectrum of pullulan-aspirin conjugate 1.

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These results have demonstrated that the unsaturated system is not destroyed during the entire course of the reaction. Broad signals of aromatic protons also suggest the covalent attachment of aromatic system onto different positions of hydroxyl groups in the pullulan polymer chains. Protons of pullulan polymer backbone/anhydroglucose unit (AGU) were detectable at 3.35–5.70 (AGU-H-1-6) ppm. The ¹H NMR spectrum has proved that samples fabricated by using the CDI method are highly pure as no sign of any impurity is detected in spectrum. The DS was also possible to be calculated by the comparison of signal intensities of the aromatic ring absorption with AGU-Hs. The DS of the samples 1–4 was found in the range of 0.32–0.40 (see Table 1).

A typical ¹³C NMR spectrum of sample 1 recorded in DMSO-d₆ shows the characteristic signals at δ 170.49 and 168.99 for the carbonyl absorptions of acetyl moiety onto aspirin and ester (CO) linkage with the polymer, respectively indicating success of the reaction (Fig. 3). It is common to observe two or three signals of C-10 of three sugars of maltotriosyl unit of the pullulan because of geometrically diverse environment. Nevertheless, C-10 absorbed at 101.84 and 95.99 ppm for pullulan-aspirin conjugate 1. The C-11-14 of pullulan maltotriose unit appears in the range of 67.5–80.03 ppm. The ¹³C NMR spectrum of pullulan-aspirin conjugate 1 is found comparable for maltotriose region with the NMR spectra of pullulan nonaacetate (Tezuka, 1998), and pullulan abietates (Hussain and Heinze, 2008).

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Figure 3 The ¹³C NMR (400 MHz, DMSO-d₆) spectrum of pullulan-aspirin conjugate 1.

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3.2 Thermal stability analysis of pullulan-aspirin conjugates

The initial decomposition (Td_i), maximum decomposition temperature (Td_m) and final decomposition (Td_f) of the pullulan-aspirin conjugate 3 were assessed by thermogravimetry (TG) and differential thermogravimetry (DTG) curves. The thermal decomposition data of pullulan-aspirin conjugate 3 are shown in Table 2 while the TG Curves and DTG thermograms of sample 3 are shown in Fig. 4.

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Figure 4 The TG (LHS) and DTG (RHS) curves of aspirin (- - -), pullulan (....) and pullulan-aspirin (^__) conjugate 3.

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The thermograms of pullulan-aspirin conjugate 3 have shown three-step degradation. The conjugate 3 was found thermally stable up to 135 °C (Td_i) with 14.2% weight loss in overall first step degradation. However Td_m and Td_f were found at 213 and 289 °C, respectively which are considerably higher than pure aspirin (152, 189 °C) indicating the extra stability imparted in pullulan-aspirin conjugate 3. The pullulan-aspirin conjugate 3 has shown major degradation in step-II. An overall weight loss of 67.68% was observed in this step. A Td_m 365 °C was observed for the conjugate 3 which is higher than Td_m of step-II for pure aspirin (327 °C). This result has indicated that aspirin becomes 38 °C more stable in conjugates. In step III, a slow degradation proceeds to completion leaving behind 0.85% char yield at 643 °C. It is concluded from above mentioned results that pullulan-aspirin conjugates are thermally more stable than pure aspirin and pullulan.

From thermogravimetric analysis, some kinetic parameters like activation energy (E_a), order of reaction (n) and frequency factor (Z) were determined. . To study the kinetic parameters (E_a and Z) of thermal degradation, Friedmann and Broido models were employed. The order of reaction n was calculated by using the Chang model. The details on the kinetics and comparison of the results are summarized in Table 2. The results have indicated comparable E_a values of the step-I degradation of conjugate 3, i.e., 34.83, 46.10 and 33.24 kJ/mol as calculated by Friedmann, Broido and Chang models, respectively. Applying same models, aspirin has shown E_a values 138, 163 and 159 kJ/mol, for the relevant first step. The n was calculated through the Kissinger model which indicated the first order kinetic of the said degradation steps. The conjugate 3 has shown E_a values of 280, 274 and 254 kJ/mol, respectively for step-II degradation, while pure aspirin showed E_a values 88, 131 and 104 kJ/mol, respectively for step-II. As all the E_a values are not very high therefore it is roughly estimated that degradation generally follows first order kinetics. It is noted from Table 2 that the application of different kinetic models on the TG curves gives almost comparable results.

3.3 Transmission electron microscopy (TEM)

The TEM analyses were carried out to study the behavior of pullulan-aspirin conjugates in solution by the solvent diffusion method. The TEM images of the molecular self-assembly have revealed the formation of nanoparticles in case of sample 3 (Fig. 5) in the range of 500–680 nm for the major population of nanoparticles. Nanoparticle formation is indicative of the fact that there lies a very good balance of hydrophobicity and hydrophilicity in sample 3 that favored the formation of nanoparticles of the water soluble biopolymer pullulan. Such nanoparticulate drug design for aspirin MPDs onto pullulan may lead to improved pharmacokinetic profile of aspirin.

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Figure 5 TEM images of the pullulan-aspirin conjugate 3 showing nanoparticle formations in the range of 500–680 nm for majority of the population.

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