Experimental evidences on the role of silica nanoparticles surface morphology on the loading, release and activity of three proteins

https://doi.org/10.1016/j.micromeso.2019.06.005Get rights and content

Highlights

  • Sol-gel procedures allow to tune the pores of silica nanoparticles for protein delivery.

  • Myoglobin, Bovine serum albumin, and Factor VIII are employed as model-proteins.

  • Loading and release data are obtained for the proteins on three silica samples.

  • The silica porosity discriminates the mechanism and the kinetics of protein release.

  • The released proteins preserve their conformation and their biological activity.

Abstract

Silica nanomaterials have been proposed as promising candidates for the delivery of drugs, including purified proteins, thus a systematic investigation on the role of silica surface porosity, for optimal protein loading and release, represents an urgent need.

Herein, we report the studies of the loading and releasing processes of different volume-proteins, namely Myoglobin, Bovine Serum Albumin and Factor VIII (FVIII), performed using three porous silica samples. To this aim, silica nanoparticles are prepared through modified sol-gel methods, in which suitable templating and swelling agents are used to enlarge the surface pores from few nm to 25 nm, but retaining the average dimensions of the nanoparticles (80 –90 nm). TEM imaging and Zeta-Potential measurements are used to characterize the silica samples.

In this study, the loading process is carried out at pH values close to the isoelectric point of each protein, and the amount of adsorbed protein is quantified and values in the range of μgprotein per mgsilica are obtained. Then, the release profiles are monitored at physiological conditions; for all systems, the release of the proteins is detected, although different behavior are observed. The release profiles are analyzed through the power law equation and through a modular exponential function to achieve information on the mechanism and on kinetics of the release profile. The data show the release from silica particles with small pores follows a diffusion-controlled mechanism, while the kinetic analysis enables to distinguish two-components associated to superficially adsorbed protein and to the portion of protein allocated inside the pores. On the other hand, the particles characterized by the largest pore size (ca. 25 nm) display non-Fickian release profile and a mono-exponential dependency, indicating a mono-modal distribution of the proteins on the silica matrix.

Moreover, the conformation or the biological activity of the released proteins has been evaluated through in-vitro methods and the data demonstrate the excellent quality of the recovered proteins.

The loading and release data were rationalized in terms of mesoporous structures of the silica nanoparticles in relation to the protein dimensions.

Introduction

The delivery of therapeutic proteins is an area of research that is engaging the attention of many researchers [[1], [2], [3]]. The interest is determined by the advantages that the treatment with proteins presents compared to conventional drugs, due to their high specificity of reaction or interaction [2]. The development of materials and methods of delivery could make the therapeutic cycles more effective and/or with optimized dosages. In particular, the use of suitable delivery materials entitle to address the present challenges of protein delivery and eventually overcome the limited stability and short half-life of the administered proteins [4]. One way to enhance the therapeutic efficiency of such proteins is to encapsulate them in a delivery system under a sustained-dosage form, with a controlled and continuous releasing rate over a period of several days [[5], [6], [7], [8]].

In the last decades, porous silica nanomaterials, have been extensively studied for pharmaceutical and medical formulations [[9], [10], [11], [12], [13]]; the use of silica nanoparticles as protein delivery systems presents several benefits: (i) the versatility and the easiness of the synthetic procedures to prepare particles with tailored morphological and surface properties [13], (ii) the long circulation time and (iii) the possibility to chemically functionalize the surface to achieve an active and efficient targeting ability [14]. Indeed, the possibility to synthesize size-controlled nanoparticles, over the range of 10 –100 nm, ensures to regulate their circulation-life and guarantees passive or active targeting thanks to an efficient permeability of the phospholipidic bilayer [13,15,16].

Both the morphology and the chemistry of nanoparticle surface play a key role in establishing and tuning the interactions with specific cargo proteins, thus affecting protein loading and releasing profile [17]. However, most of the work has been focused on the loading and delivery of small size proteins [2,12,13] and only few reports have investigated large and/or therapeutic proteins [18,19]. The data indicate that, specifically when the loading of large proteins is desirable, a careful consideration of the particle superficial morphology and the pore size are necessary [18,19]. Omar et al. reported the preparation of an ultra-large pore mesoporous silica, using a two-step method based on a seed-mediated approach where iron oxide nuclei acted as seed; these particles were able to load and release the protein mTFP-Ferritin (ca. 534 KDa) [18]. Using a similar preparation method, Kwon et al. reported the use of an extra-large pore mesoporous silica for cytokine delivery [19]. To broaden the use of mesoporous silica for the transfer of large protein, the presence of iron oxide worthless for subsequent cargo delivery.

More recently, Clemments et al. [20] mapped the distribution of three different proteins (with molecular weights of 17 kDa, 66 kDa and 138 kDa) onto mesoporous silica nanoparticles with 3 or 6 nm-pores; they showed that the penetration depth of the proteins within the porous structure of the inorganic matrix strongly depends either on pore size and protein volume.

Moreover, although it has been proven that the presence of large pores is beneficial for bulky biomolecules loading, it is still unclear whether these may become counterproductive for the controlled release of proteins. To the best of our knowledge, systematic and comparative studies on porous silica nanomaterials characterized by different surface morphologies, which address the loading, stability and release efficiencies of proteins of different sizes are limited. Instead, the possible exploitation of silica colloids as carriers needs to fully understand which characteristics of the matrix determine the controlled loading and release of therapeutic proteins. Moreover, so far, minimal information is also available on the configuration and/or activity of the released proteins, a parameter that have great impact on protein functions.

In the present work, we report the results of a systematic investigation on the impact of silica nanomaterial morphologies on the loading and release of small, medium and large size model proteins.

Section snippets

Materials

Factor VIII (FVIII) was a kind gift from Bayer Italia. Myoglobin from equine skeletal muscle (MG, lyophilized powder 95–100%), bovine serum albumin (BSA, lyophilized powder ≥96%), tetraethylorthosilicate (TEOS, 98%), hexadecyltrimethylammonium bromide (CTAB, 95%), hexadecyltrimethylammonium p-toluensulfonate (CTATos), triethanolamine (TEAH3, >98%), mesitylene (98%), hydrochloridric acid (HCl, 37%), sodium hydroxide (NaOH, pellets 99%), potassium phosphate monobasic (KH2PO4, ≥99.0%), ammonium

Preparation and characterization of different porous silica nanoparticles

Silica nanoparticles with different pore size were prepared and systematically used to test the role of silica nanoparticles morphology on protein adsorption and release efficiencies. To this end, the Stöber methodology has been modified by adding suitable surfactants with the capabilities to act as templating agents; it has been demonstrated that the addition of templating agents in the synthetic procedure is able to originate a pore network on the silica colloids and the dimensions of the

Conclusion

In this work, porous silica nanoparticles with different pore size have been prepared and used to evaluate the role of surface morphology on protein loading and releasing behavior. To this aim small-, medium- and large-volume proteins have been employed, in order to investigate the correlation between the carrier type and the size of the protein.

Overall, our results highlight that the use of silica system with proper morphology allows the suitable allocation of the protein, thus leading to an

Acknowledgment

The Università degli Studi di Perugia and the Ministero per l’Università e la Ricerca Scientifica (MIUR - Rome) are thanked for the financial support through the program “Dipartimenti di Eccellenza 2018–2022” (grant AMIS). F.F. acknowledges the National Multiple sclerosis society (Grant: PP-1603-08205) and Telethon (Grant: GGP17094) for financial support.

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