Preparation and Preliminary Evaluation of Povidone Single-Chain Nanoparticles as Potential Drug Delivery Nanocarriers

One of the key areas in nanomedicine is the use of nanometer-sized materials as nanocarriers for therapeutic and diagnostic (theranostic) purposes. In particular, nanoparticles (NPs) have attracted a considerable attention due to their small size that confers the ability to be transported more easily through the body. Ideally, nanocarriers would be biocompatible and biodegradable so the involvement of soft matter-based NPs is an interesting approach. Folding individual polymer chains to single-chain nanoparticles (SCNPs) endows the resulting soft nano-objects with promising prospects for drug encapsulation and subsequent controlled delivery. In this work, we report on the preparation and preliminary (in vitro) evaluation of Povidone SCNPs as potential drug delivery nanocarriers. We select Povidone (polyvinylpyrrolidone) as a water-soluble polymer with a large commercial use in medicine, which is biocompatible and non-antigenic as well as safe for oral and topical applications. For evaluation of Povidone SCNPs as drug delivery nanocarriers, we select two drugs with reported anti-cancer activity: (i) Cisplatin, a widely used hydrophilic anticancer agent for treatment of a variety of cancer cells; and (ii) Lovastatin, a lipophilic compound with in vitro anti-proliferative, pro-apoptotic and anti-invasive effects in different cancer cell lines. After showing release of these drugs from Povidone SCNPs, we demonstrate that these nanoparticles can be rendered fluorescent in combination with functional aggregation-induced emission (AIE) fluorophore molecules paving the way to the potential development of theranostic Povidone SCNPs. Open Access Received: 23 May 2019 Accepted: 08 July 2019 Published: 10 July 2019 Copyright © 2019 by the author(s). Licensee Hapres, London, United Kingdom. This is an open access article distributed under the terms and conditions of Creative Commons Attribution 4.0 International License. Med One. 2019;4:e190013. https://doi.org/10.20900/mo.20190013


INTRODUCTION
According to the European Commission (EC) recommendation 2011/696/EU, nanomaterial is defined as a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range of 1 nm to 100 nm (1 nm = 0.001 µm = 10 −9 m) [1].
Concerning the use of NPs in nanomedicine, the biological transport performed anatomically into cellular and sub-cellular levels is severely affected by NP's physical and chemical attributes [7]. In this way, NPs have to overcome several obstacles/barriers (pH, cell membrane, pores, chemical or enzymatic reactions…) to reach their target. One of the key areas in nanomedicine is the use of nanometer-sized materials as nanocarriers for diagnostic and therapeutic (theranostic) purposes [8].
NPs' physical and chemical attributes including size, shape, flexibility, hydrophobicity, etc., are critical for a successful theranostic achievement.
One of the critical points to overcome when drug target is located intracellularly is drug passage through the cell membrane [9]. between 0.25 to 3 μm that have been previously a foreign body [10].
Non-phagocytic clathrin-mediated endocytosis is another way of internalization, which may be performed by ligands that are bound to nanoparticle receptor and an invagination process happens to introduce NPs with a size between 120 and 200 nm [11]. Finally, nanoparticles can also be introduced into the cellular compartment by calveolae-mediated endocytosis, i.e., through invaginations present in membrane by pores below 80 nm [12].
Ideally, nanocarriers would be biocompatible, biodegradable, and specifically designed to obtain an appropriate bio-distribution.
Researchers have been able to synthesize a variety of NPs loaded with drugs that can be observed by imaging methods (e.g., magnetic resonance imaging (MRI) [13], computed tomography (CT) [14], positron emission tomography (PET) [15], fluorescence microscopy [16]). Among the potential advantages that could be achieved using NPs loaded with therapeutic molecules we can mention: (i) improved physical and chemical stability of drug; (ii) increased absorption of drugs; (iii) controlled release of drugs; (iv) superior crossing of tissue/cell-specific barriers to penetrate into cells; and (v) reduced adverse effects and toxicity associated with the administration of free drugs. However, the main concern of NPs in nanomedicine is their potential toxicity for living systems, mainly derived from their accumulation in different tissues and organs [17]. In this way, using soft matter-based nanocarriers seems to be a particularly interesting solution.
In this work, we report on the preparation and preliminary (in vitro) evaluation of Povidone single-chain nanoparticles (SCNPs) as potential drug delivery nanocarriers (see Figure 1). Polyvinylpyrrolidone, also known as Povidone or PVP, is a highly polar, amphoteric polymer used in the pharmaceutical field as an excipient, as well as in the cosmetic, food and textile industries [18]. PVP is biocompatible and non-antigenic, safe for oral and topical applications, and it is easily dispersible in water. Its conjugation with iodine forms the active ingredient PVP-iodine, largely used as antiseptic (e.g., Betadine). We hypothesized that by folding individual PVP chains at high dilution via intra-chain cross-linking [19], the resulting Povidone SCNPs will be endowed with local domains (pockets) to which drugs can be temporary immobilized for their subsequent controlled delivery. Previously, we and others have demonstrated the possibilities that SCNPs offer for the construction of responsive drug delivery nanocarriers [20][21][22]. A detailed analysis of recent findings in the field of drug delivery from SCNPs can be found in a relevant review paper by Kröger and Paulusse [23]. Following a previous recent investigation [24], we will perform the folding of PVP chains through radical generation in the pyrrolidone units via Fenton reaction followed by intra-chain radical-radical coupling events. For evaluation of Povidone SCNPs as potential drug delivery nanocarriers, we selected two drugs with reported anti-cancer activity [25,26]: (i) Cisplatin, a widely used hydrophilic anticancer agent that has been shown to be highly effective in the treatment of testicular, ovarian, breast, bladder, lung, head and neck cancers; and (ii) Lovastatin, a lipophilic compound which has demonstrated to exert anti-proliferative, pro-apoptotic and anti-invasive effects in different cancer cell lines with varying sensitivity. After showing the drug delivery characteristics of Povidone SCNPs as nanocarriers, we will turn our attention to the possibility to render fluorescent the Povidone SCNPs. Toward this end, we select a new kind of fluorophore molecules which are opposite to conventional ones, typically affected by the well-known and deleterious aggregation-caused quenching (ACQ) effect [27]. Hence, we investigated the use of a functional aggregation-induced emission (AIE) fluorophore [28] to introduce diagnostic characteristics into the Povidone SCNPs.

Fluorescence spectroscopy
Photoluminiscence (PL) spectra were recorded at room temperature on an Agilent Cary Eclipse spectrometer (USA) at an excitation wavelength of 365 nm.

Synthesis of Povidone Single-Chain Nanoparticles (SCNPs)
Povidone SCNPs were synthesized following the method reported in [24]. In brief, PVP-based SCNPs were produced at room temperature by mixing two solutions: solution A containing Povidone dissolved in water (50 mg, 100 mL, 0.5 mg/mL) and H2O2 (113 μL, 10 mM), and solution B containing Povidone dissolved in water (same concentration and amount as solution A) and FeCl2 (63.4 mg, 5 mM). The pH of the mixed system was adjusted at pH = 3.5 with acetic acid (0.1 M). After reaction completion, the mixture was dialyzed for 24 h in order to remove traces of unreacted FeCl2. Finally, the system was freeze-dried and Povidone SCNPs were obtained as white powders.

Preparation of Cisplatin-Loaded Povidone SCNPs
First, a solution of Cisplatin (CP) in water (5 μg/mL, containing 0.9% of NaCl) was prepared. Then, 1.5 mg of Povidone SCNPs were dissolved in 5 mL of the solution containing CP and the mixture was incubated for 24 h in the dark at room temperature. In order to purify the CP-loaded Povidone nanocarriers, extraction with 5 mL of CHCl3 was carried out (this process was repeated twice); CHCl3 was evaporated using a continuous argon stream, and the CP-loaded Povidone nanocarriers were isolated.

Cisplatin Drug Delivery from Povidone SCNPs
The delivery of cisplatin from CP-loaded Povidone SCNPs was where CPt is the value of absorbance at 265 nm measured at time t (h), CPt=0 is the value of absorbance measured at the beginning of the experiment, and CPt=96h is the value of absorbance measured after 96 h.

Preparation of Lovastatin-Loaded Povidone SCNPs
where LOt is the value of absorbance at 288 nm measured at time t (h), LOt=0 is the value of absorbance measured at the beginning of the experiment, and LOt=2.5h is the value of absorbance measured after 2.5 h.

Preparation of Fluorescent Povidone SCNPs
Povidone SCNPs were dissolved in water at a concentration of

Preparation and Size Characterization of Povidone SCNPs
Following a recent investigation [24], we performed the folding of Povidone chains through radical generation in the pyrrolidone units via Fenton reaction followed by intra-chain radical-radical coupling events (see Figure 1 and ref. [24] for details). The synthesis process was performed by starting from a commercially available water-soluble PVP homopolymer and by using reactive oxygen species (ROS) generated via Fenton reaction for the intra-chain folding/collapse process of individual polymer chains.
Hydroxyl radical is the most reactive radical among the reactive oxygen species. Its reaction with Povidone is expected to generate macroradicals centered in three possible positions, taking into account the labiality of the hydrogen atoms present in its structure (see Figure 2) [29]. In general, the rate of hydrogen abstraction is dependent on the dissociation energy of the X-H bond to form the radical. Basic thermodynamic calculations show that C-H bonds α-positioned to a heteroatom or a C=O group are lower in energy, mainly due to the stabilization of the radical product [30]. Within the three C-H bonds shown in Figure 2, the most favorable PVP macro-radical expected via

Experimentally, the intra-chain cross-linking of the individual (linear)
Povidone chains to single-chain nanoparticles was performed in water, at room temperature in the presence of hydroxyl radicals (generated by Fenton reaction) and at very dilute conditions (0.5 mg/mL) in order to avoid inter-molecular cross-linking events. Figure 3 illustrates the Taken together, the above SEC, DLS and SAXS data confirm the significant chain compaction that takes place upon folding/collapse of individual Povidone linear chains to SCNPs by means of radical generation in the pyrrolidone units via Fenton reaction followed by intra-chain radical-radical coupling events. We hypothesized that the resulting Povidone SCNPs will be endowed with local domains (pockets) to which drugs can be temporary immobilized for their subsequent controlled delivery [35].

Nanocarriers
For evaluation of Povidone SCNPs as potential drug delivery nanocarriers, we selected two drugs with reported anti-cancer activity: (i) Cisplatin, a widely used hydrophilic anticancer agent for treatment of a variety of cancer cells [24]; and (ii) Lovastatin, a lipophilic compound with in vitro anti-proliferative, pro-apoptotic and anti-invasive effects in different cancer cell lines [26].

Cisplatin (CP)
Povidone SCNPs were loaded with Cisplatin following the procedure essentially identical to those of the neat Povidone SCNPs (i.e., hydrodynamic diameter ≈ 11 ± 3 nm; ζ-potential ≈ −0.025 ± 0.03 mV). Figure 5A shows the controlled delivery of CP from Povidone SCNPs to water as monitored by UV-Vis spectroscopy for a period of 96 h. Over time, a clear UV-Vis absorption band develops centered at ~265 nm which is due to the progressive release of Cisplatin to the water solution. The corresponding in vitro drug delivery curve obtained from the data reported in Figure 5A is illustrated in Figure 5B. These results indicate that CP-loaded Povidone SCNPs have a great potential as nanocarriers since they provide with a sustained, controlled release of Cisplatin that amounts to ~28% in 2 h, ~75% in 24 h, and ~95% in 48 h. These results are in agreement with previous studies concerning the ability of SCNPs to function as drug delivery agents [20][21][22]. Nevertheless, before practical use of CP-loaded Povidone SCNPs a more complete characterization that is out of the scope of the present paper should be performed (e.g., accurate determination of drug loading content and encapsulation efficiency, evaluation of toxicity issues, effect of pH and temperature).

Lovastatin (LO)
Povidone SCNPs were loaded with Lovastatin following the procedure described in Materials and Methods section. Since LO, which is a lipophilic compound, is almost insoluble in water we monitor the release of this drug to a solvent in which LO is highly soluble, such as DMF. This is a very simple model of LO delivery to lipophilic tissues. Figure 6A  from the data reported in Figure 6A is illustrated in Figure 6B. These results indicate that Povidone SCNPs can be also used as nanocarriers of lipophilic drugs since they provide with a relatively fast release of Lovastatin to DMF that amounts to 63% in 1 h, and near 100% in 2 h.
However, more realistic models are required to validate these preliminary results, including cellular and toxicity studies as well as a complete characterization of drug loading content and encapsulation efficiency.

Endowing Povidone SCNPs with Fluorescent Properties
Fluorophore molecules can be monitored by fluorescence spectroscopy and microscopy, so they have long been used in biomarker analysis, immunoassays, and diagnostic imaging, including cancer diagnosis [36]. However, one of the main limitations of conventional fluorophore molecules is which often suffer from the well-known (and deleterious) aggregation-caused quenching (ACQ) effect [27]. In recent years, a new class of molecules (aggregation-induced emission (AIE) fluorophores) has been discovered that are nearly non-emissive molecules when molecularly dissolved in a solvent but become highly fluorescent when aggregate or after being molecular immobilized due to restriction of the intramolecular rotation in the aggregate/immobilized state leading the excitations to decay radiatively [37,38]. In this work, we evaluate the ability of an AIE fluorophore (TPE-BA, see the chemical structure in Figure 7A) to endow Povidone SCNPs with fluorescent properties. As illustrated in Figure 7B Interestingly, a water solution containing both Povidone SCNPs and TPE-BA molecules shows a 5-fold increase in fluorescence (see Figure 7B) and

CONCLUSIONS
In summary, a strategy has been developed toward theranostic SCNPs based on Povidone, a water-soluble polymer with a large commercial use in medicine, which is biocompatible and non-antigenic as well as safe for oral and topical applications. Povidone SCNPs were prepared at high dilution through radical generation in the pyrrolidone units via Fenton reaction followed by intra-chain radical-radical coupling events.