Metal-free thermally-responsive pseudohybrid nanoparticles based on 2-hydroxypropyl-β-cyclodextrin

Abstract

Pseudohybrid nanoparticles of 800 nm in diameter based on self-assembled high-substituted 2-hydroxypropyl-β-cyclodextrin fabricated in the presence of iron(II) salts were found to be thermally responsive within narrow range of temperature. As-prepared metal-free nanoparticles are stable in aqueous solution at room temperature. However, the increase in temperature results in break-like collapse of nanoparticles to yield species of 400 nm in diameter. On the example of phenolphthalein, it was shown that nanoparticles loaded with model drug decompose to release superficial guest-host inclusion complexes. Local hyperthermia provokes nanoparticles decomposing and drug releasing that allows recognizing focuses of pathology in human body. It might be used for diagnostic medicinal aims and be also considered as basis for the construction of intravascular drug delivery/release systems.

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Introduction

In recent years, controlled drug delivery has become one of the most interesting branches in therapeutic medicine.^1–6 Hybrid nanoparticles based on biodegradable polymers and magnetic cores are used as containers for drugs.^7–12 In most cases magnetic metal oxides (Fe₃O₄, γ-Fe₂O₃) conform to cores of hybrid nanoparticles to fabricate magnetically responsive species for drug delivery.

Targeting migration of as-prepared magnetic nanoparticles within human body apparently needs high-power magnetic fields to overcome drastic hydrodynamic liquid resistance and is difficult enough to be realized. The following drug release needs heating, e.g., by high-frequency magnetic field.¹³ In this case, thermal degradation of nanospecies leads undoubtedly to coagulation of metal oxide cores, which should be removed. So, it is more reasonable to use metal-free nanoparticles for the drug delivery throughout human body.

A tendency of cyclodextrins (CDs) for self-assembling in aqueous solutions is worth growing attention due to opportunity to fabricate advanced nanosystems.^14–17 There are a lot of applications (supramolecular chemistry, biology, medicine, pharmaceutics, drug delivery and so on) for efficient use of these nontoxic “green” materials.^16–21

Substituted β-CDs, e.g.,2-hydroxypropyl-β-cyclodextrin (HP-β-CD) were found to exhibit striking properties in solutions. These compounds not only provide their cavities for a wide variety of “guest” molecules but also tend for self-assembling to form nanoparticles of different morphology and size distribution. High-substituted HP-β-CD was reported to form macromolecular nanospecies in aqueous solutions of iron(II) salts.²²

High-substituted HP-β-CD forms nanoparticles of two types depending on molar ratio of iron(II) ions and HP-β-CD.²² The hybrid nanoparticles containing iron oxide core were isolated from aqueous solution in the presence of 10-fold excess of iron(II) ions. The second type of the nanoparticles forms in 100-fold excess of iron(II) ions and presents iron-free (pseudohybrid) species. These pseudohybrid nanoparticles are non-magnetic and exhibit more solubility than hybrid nanoparticles in aqueous solutions.

The heat treatment is known to be used in order to collapse nanoparticles and to release drugs.^23–27 Nanoparticles based on β-CD-modified magnetic Fe₃O₄ were found to decompose and release drug molecules by heat treatment by on–off switching of high-frequency magnetic field.²³

However, there are three disadvantages in this case. The first one is the need to use “external” source of the heat. The second one is the fact that the drug release should take place exactly in focus of pathology to get the highest effect. The third one is the need to use very high temperatures in order to decompose magnetic nanoparticles. In the work cited, temperature of decomposition was 42–45 °C what is high enough for biologic materials.

Meanwhile, the heat treatment by “external” source, e.g., high-frequency magnetic field is evident to impact unselectively the compounds. In particular, it is important for human body in which magnetic field itself may provoke pathologic processes.

On the other hand, it is difficult enough to deliver magnetic nanoparticles intentionally within the human body by means of “external” magnetic field.

This work reports on a different approach to the challenge of the drug delivery and drug release by means of non-magnetic metal-free nanoparticles based on self-assembled HP-β-CD. Drug release is based on a thermal hypersensitivity of these species in aqueous solutions at 37 °C.

Results and discussion

As-prepared nanoparticles are iron-free species of 800–840 nm in diameter and seemed to be extremely thermally sensitive. It is of interest that although HP-β-CD self-assembling was detected in solutions only in the presence of iron(II) salts, the nanoparticles based on HP-β-CD were found to comprise no iron oxides and contained only traces of iron (0.1 wt%).

Whereas no metal oxide cores are present within the as-prepared nanoparticles,²² the species obtained are sure to be pseudohybrids. The pseudohybrid supramolecular species based on HP-β-CD were also found to trap a minority of mineral salts (sulfates, halides) and to be well soluble in water.

Average radii of effective hydrodynamic spheres (R_h) for the pseudohybrid nanoparticles based on self-assembling HP-β-CD were found to depend unambiguously on temperature of the synthesis. Fig. 1 presents a plot of R_h as a function of temperature of the synthesis. To begin with 37 °C the increase in temperature of the synthesis leads to a decrease in hydrodynamic radius of the particles obtained. HP-β-CD nanoparticles fabricated in solution at room temperature were detected to be the largest ones having 840 ± 40 nm in diameter whereas those synthesized at 43 °C are much smaller (230 ± 10 nm). Marked difference in the dimensions of the nanoparticles prepared under various temperature conditions is indicative of their non-covalent nature and strong thermal sensibility. It was shown by the special experiments the radius of hydrodynamic sphere of nanoparticles do not depend on the material concentration in the range of 0.01–1 wt% in water solution and do not depend on pH in the range of 5–9.5.

Fig. 1 Radii (R_h) of effective hydrodynamic spheres of pseudohybrid nanoparticles based on self-assembling HP-β-CD (0.3 wt%) vs. temperature of synthesis in aqueous solutions (pH 6.7).

The pseudohybrid nanoparticles being quite stable in solutions at room temperature were found to be thermally responsive. Fig. 2 shows plot of hydrodynamic radii of pseudohybrid nanoparticles in aqueous solutions as function of temperature.

Fig. 2 Radii (R_h) of HP-β-CD pseudohybrid nanoparticles (0.3 wt%) fabricated at 25 °C vs. in aqueous solutions (pH 6.7): heating (1), cooling (2).

Hydrodynamic radii of the pseudohybrid nanoparticles are seen to be constant up to 37 °C. To begin with this temperature, a striking collapse is observed with the increase in the solution temperature to yield smaller nanoparticles. It is surprising that the partial decomposition of the species takes place within the range of 1–2 grads. So, 37 °C is critical temperature for the metal-free nanoparticles based on the self-assembled HP-β-CD.

The cooling of the solution after being heated up to 44 °C leads to a slight increase in the average hydrodynamic radii of the pseudohybrid nanoparticles. However, no former dimension of the nanoparticles was achieved at 25 °C (Fig. 2).

It is reasonable that the heating of the solution favors decomposition of the pseudohybrid nanoparticles formed by non-covalent bonding. It is really striking that the partial destruction of the species is observed within narrow interval at 37 °C, which is close to human body's temperature.

Break-like change in dimensions of the pseudohybrid nanoparticles is probably to result from cleavage of non-covalent hydrogen bonds between HP-β-CD molecules throughout the nanoparticles.

We have found that the above self-assembling of HP-β-CD at 43–44 °C results in the formation of smaller nanoparticles of 220 ± 10 nm in diameter. This result indicates that the increase in temperature inhibits the HP-β-CD self-assembling.

The metal-free nanoparticles based on HP-β-CD obtained at higher temperature (43 °C) exhibit the same thermal sensitivity. The results are presented in Fig. 3.

Fig. 3 Radii (R_h) of effective hydrodynamic spheres of pseudohybrid nanoparticles based on self-assembling HP-β-CD vs. temperature for pseudohybrid nanoparticles (0.3 wt%) obtained at 43 °C in aqueous solutions (pH 6.7): heating (1), cooling (2).

Visualization of nanoparticles obtained before and after heating was carried out by means of transmission electron microscope (TEM). It was shown that the nanoparticles obtained at 25 °C have a spherical form (Fig. 4(a)). The average radius of these particles is 300 nm. This size correlates with the results of DLS in which the average hydrodynamic diameter of pseudohybrid nanoparticles equaled to 400 nm.

Fig. 4 Electron microphotographs of pseudohybrid nanoparticles fabricated at 25 °C before (a) and after (b) incubation of solution at 43 °C. The concentration of nanoparticles was 0.1 wt%.

The heat treatment leads to decrease of nanoparticles radius from 300 nm to 80 nm (Fig. 4(b)). This result correlates with the average hydrodynamic radius of heated pseudohybrid nanoparticles equaled to 120 nm. Because of swelling in aqueous solution, the radii measured by DLS were slightly larger than the sizes obtained microscopically.

Additional information about the behavior of investigated pseudohybrid nanoparticles in aqueous solutions can be obtained by means of static light scattering (SLS).^28–31 Relative light scattering intensity C_w/(I−I_solv) (C_w is weight concentration of nanoparticles, I and I_solv are light scattering intensities of solution and solvent, respectively) for the pseudohybrid nanoparticles obtained at 25 °C was observed to exhibit a striking drop by heat treatment within the range of 34–37 °C. Fig. 5 shows the feature at 34–37 °C just before a partial decomposition of the pseudohybrid nanoparticles (Fig. 2 and 4(a)). Irregular change in the relative light scattering intensity C_w/(I−I_solv) for the pseudohybrid nanoparticles obtained at 25 °C is indicative of critical rearrangement of the metal-free nanoparticle shell.

Fig. 5 Plot of relative light scattering intensity C_w/(I−I_solv) for HP-β-CD pseudohybrid nanoparticles (0.3 wt%) fabricated at 25 °C vs. solution temperature: heating (1), cooling (2).

On contrary, the relative light scattering intensity increases monotonically by cooling of the solution from 43 °C to 25 °C (Fig. 6).

Fig. 6 Plot of relative light scattering intensity C_w/(I−I_solv) for HP-β-CD pseudohybrid nanoparticles fabricated at 43 °C vs. solution temperature: heating (1), cooling (2); concentration of pseudohybrid nanoparticles C_w is 0.3 wt%.

On the other hand, there are no features in the relative light scattering intensity C_w/(I−I_solv) for the pseudohybrid nanoparticles obtained at 43 °C. The data is presented in Fig. 6.

Decrease in dimensions of the pseudohybrid nanoparticles based on HP-β-CD is accompanied by appearance of free HP-β-CD molecules in solution of nanoparticles. HP-β-CD is known to form inclusion compounds with guest molecules, e.g., phenolphthalein.³² However, only intramolecular ring-closed colorless form of phenolphthalein may be included in cavities of free HP-β-CD molecules.

There are steric hindrances for red-colored “open” structure of phenolphthalein to be trapped by free HP-β-CD.³² Red-colored borate buffer solution of phenolphthalein is decolorized in the presence of HP-β-CD due to shifting of equilibrium towards colorless form of phenolphthalein, which are trapped by HP-β-CD cavities as shown in Fig. 7.^32–34

Fig. 7 Phenolphthalein–HP-β-CD complex.

The plots of absorbances of the solutions of the pseudohybrid nanoparticles measured at 553 nm (D₅₅₃) vs. phenolphthalein concentration are shown in Fig. 8. According to spectrophotometric data, there is a difference between titration curves of borate buffer solutions of the pseudohybrid nanoparticles before and after heat treatment (curves 2 and 4, respectively). The data obtained indicates that heat treatment of the solution of the pseudohybrid nanoparticles at 46 °C results in appearance of free HP-β-CD molecules. For the comparison, the plots of phenolphthalein and free HP-β-CD as functions of phenolphthalein concentration are also shown in Fig. 8 (curves 1 and 3, respectively).

Fig. 8 Absorbance of pure buffer borate solution (1), 1 wt% solution of pseudohybrid nanoparticles (2), 1 wt% solution of HP-β-CD (3) and 1 wt% solution of pseudohybrid nanoparticles after thermal treatment at 46 °C (4) vs. phenolphthalein concentration; pH 9.2, λ = 553 nm.

It is obvious that the spectrophotometric data permits the explanation of the results of DLS measurements (Fig. 2). The decomposition of the pseudohybrid nanoparticles observed at 37 °C leads to the increase in number of centers capable to forming complexes. The titration curve for the solution of the pseudohybrid nanoparticles after heat treatment at 46 °C coincides with the curve obtained for free HP-β-CD.

UV-VIS absorption spectra of phenolphthalein in borate buffer solutions (pH 9.2) in the presence of pseudohybrid nanoparticles can be an evidence of complex formation. The data are shown in Fig. 9. The addition of pseudohybrid nanoparticles (curves 2, 3, 4) to phenolphthalein solution leads to decrease in the absorbance of initial dye (curve 1).

Fig. 9 The absorption UV-VIS spectra for pure phenolphthalein (1) and phenolphthalein in the presence of pseudohybrid nanoparticles fabricated at 25 °C with concentrations: 0.1% wt (2); 0.15% wt (3); 0.8% wt (4). Concentration of phenolphthalein, C = 4.3 × 10⁻⁵ M, buffer borate solutions (pH 9.2).

Thus, the UV-VIS absorption spectra suggest unambiguously that there is a complex formation of pseudohybrid nanoparticles with phenolphthalein.

The result obtained is in accordance with assumption that only surface HP-β-CD molecules of pseudohybrid nanoparticles, the cavities of which are open towards the solution, form phenolphthalein adducts.

Cytotoxicity of metal-free nanoparticles based on HP-β-CD and initial HP-β-CD was investigated on the example of cells of adenocarcinoma of MCF-7/R⁻¹ human mammary gland. Plot of survival of the cells vs. concentration of nanoparticles is presented in Fig. 10. The results obtained are indicative of nontoxicity of pseudohybrid nanoparticles based on self-assembling HP-β-CD up to 0.2 wt% concentrations.

Fig. 10 Plot of survival of MCF-7/R⁻¹ cells vs. concentration of HP-β-CD (1) and pseudohybrid nanoparticles based on self-assembling HP-β-CD (2).

So, according to the data presented, the HP-β-CD nanoparticles may be used as effective drug carriers, which possess the capacity for recognizing focuses of pathology and releasing drugs due to heat treatment within hyperthermia focuses.

Experimental procedures

2-Hydroxypropyl-β-cyclodextrin (relative molar mass is 1540, substitution degree is 6.8) and phenolphthalein purchased by Merk. The pseudohybrid nanocomposite were obtained at the reaction of (NH₄)₂Fe(SO₄)₂ with NaH₂PO₂ in the presence of HP-β-CD (degree of substitution is 6.8) in alkaline aqueous solutions (pH 13). The molar ratio of the components (HPCD : (NH4)₂Fe(SO₄)₂ : NaH₂PO₂) is 1 : 100 : 100. It was estimated the opportunity of self-assembling of HP-β-CDs under the same conditions.

It leads to formation of stable water soluble macromolecular compounds. Syntheses were carried out at 25, 29, 35, 39, 41, 43, and 45 °C. The high molecular weight products were separated from low molecular substances by means of dialysis. Membranes “Sigma-Aldrich” (USA) with MWCO 15000 were used for dialysis of pseudohybrid nanoparticles against pure water during 4 h. Water was changed 8 times.

According to elemental analysis all obtained materials comprise only traces of iron (less 0.1%).²²

DLS and SLS measurements were carried out at scattering angle of 90° using DynaLS photon correlation spectrometer in thermostatic cells. Linearly polarized light of He–Ne laser (wavelength is 632.8 nm) was used. Hydrodynamic radii were calculated from the diffusion coefficients according to the Stokes–Einstein equation. The hydrodynamic radii were shown to be independent of the light scattering angle. The concentration of pseudohybrid nanoparticles (C_w) was defined as a relation of pseudohybrid nanoparticles mass to solution mass multiplied on 100%.

For the electron-microscopic examination the suspension of composite dispersion at a concentration was applied. The concentration of suspension was 0.2 g l⁻¹. This suspension was contrasted by phosphotungstic acid and analyzed by microscope JEOLJEM-1400 (JEOL). TEM images was obtained by OSIS camera and processed by computer technique OSIS iTEM.

UV-VIS spectra were obtained on Ultraspec-4050 spectrophotometer (LKB, Sweden). Titration by phenolphthalein was performed in borate buffer solution (pH 9.2) and absorbance of solution was measured at 553 nm in thermostatic cells.

Cytotoxicity of pseudohybrid nanoparticles based on self-assembling HP-β-CD was studied for cells of adenocarcinoma of MCF7/R according to work.³⁵ The cells containing pseudohybrid nanoparticles in serumless medium were incubated for 1 h. Then the serumless medium was replaced with total growth one followed by addition of 10 wt% embryonic serums. The as-prepared cells were incubated in 5% CO₂ atmosphere at 95% humidity for 3 h. The error interval was not more than 2% for each data point.

Determination of the number of cells alive was carried out according to the method based on the capacity of tetrazolium-blue dye for reducing within mitochondria to yield water insoluble violet product (formazan).

The numbers of live cells are proportional to formazan quantity detected spectrophotometrically.³⁶ The absorbance of the alveoli solution with cells was measured at 550 nm using Multiscan photometer (“Titertek”, USA). The numbers of live cells (N) were calculated by dividing of absorbance of alveolar solution with given concentration by absorbance of control solution. Each value of N was calculated as arithmetic mean from 3 measurements.

Acknowledgements

Authors thank Nikolay S. Melik-Noubarov for providing cytotoxicity measurements.

Notes and references

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