A New Methodology to Create Polymeric Nanocarriers Containing

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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A New Methodology to Create Polymeric Nanocarriers Containing Hydrophilic Low Molecular-Weight Drugs: A Green Strategy Providing a Very High Drug Loading María Gabriela Villamizar-Sarmiento,†,‡,§ Elton F. Molina-Soto,‡ Juan Guerrero,∥ Toshimichi Shibue,⊥ Hiroyuki Nishide,# Ignacio Moreno-Villoslada,*,‡ and Felipe A. Oyarzun-Ampuero*,†,§ †

Department of Sciences and Pharmaceutical Technology, University of Chile, Santiago de Chile 8380494, Chile Instituto de Ciencias Químicas, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia 5110033, Chile § Advanced Center for Chronic Diseases (ACCDiS), Santiago 8380494, Chile ∥ Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40 Santiago 9170124, Chile ⊥ Materials Characterization Central Laboratory; School of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan # Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku, Tokyo 165-8555, Japan

Downloaded by UNIV OF SOUTHERN INDIANA at 21:17:18:241 on June 03, 2019 from https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.9b00097.

‡

S Supporting Information *

ABSTRACT: To date, a large number of active molecules are hydrophilic and aromatic low molecular-weight drugs (HALMD). Unfortunately, the low capacity of these molecules to interact with excipients and the fast release when a formulation containing them is exposed to biological media jeopardize the elaboration of drug delivery systems by using noncovalent interactions. In this work, a new, green, and highly efficient methodology to noncovalently attach HALMD to hydrophilic aromatic polymers to create nanocarriers is presented. The proposed method is simple and consists in mixing an aqueous solution containing HALMD (model drugs: imipramine, amitriptyline, or cyclobenzaprine) with another aqueous solution containing the aromatic polymer [model polymer: poly(sodium 4-styrenesulfonate) (PSS)]. NMR experiments demonstrate strong chemical shifting of HALMD aromatic rings when interacting with PSS, evidencing aromatic− aromatic interactions. Ion pair formation and aggregation produce the collapse of the system in the form of nanoparticles. The obtained nanocarriers are spheroidal, their size ranging between 120 and 170 nm, and possess low polydispersity (≤0.2) and negative zeta potential (from −60 to −80 mV); conversely, the absence of the aromatic group in the polymer does not allow the formation of nanostructures. Importantly, in addition to high drug association efficiencies (≥90%), the formed nanocarriers show drug loading values never evidenced for other systems comprising HALMD, reaching ≈50%. Diafiltration and stopped flow experiments evidenced kinetic drug entrapment governed by molecular rearrangements. Importantly, the nanocarriers are stable in suspension for at least 18 days and are also stable when exposed to different high ionic strength, pH, and temperature values. Finally, they are transformable to a reconstitutable dry powder without losing their original characteristics. Considering the large quantity of HALMD with importance in therapeutics and the simplicity of the presented strategy, we envisage these results as the basis to elaborate a number of drug delivery systems with applications in different pathologies. KEYWORDS: nanocarriers, nanomedicines, aromatic-aromatic interactions, tricyclic drugs, hydrophilic drugs

1. INTRODUCTION To date, a large number of the available drugs show low molecular-weight ( 0.997) in the range of concentrations between 7 × 10−5 and 7 × 10−7 M (molar extinction coefficient was 12 712 M−1 cm−1). Blank experiments dialyzing free AMT were carried out at the same conditions.

of the NPs with spectrophotometry through instantaneous turbidity changes. 2.2.5. Loading in the Nanocarriers Containing HALMD. The drug loading (% w/w) was calculated by dividing the amount of drug associated in the formulation by the total weight of the nanocarriers. The drug content into the NPs was calculated indirectly by quantifying the non-interacting drug in the medium; the separation of nanocarriers and free drug was done by using Vivaspin 6 tubes (MWCO 3 kDa, 5000g × 40 min). The quantification of the drug (AMT) was done by measuring the absorbance at 239 nm (Agilent 8453 spectrophotometer, USA). For the calculation of the total weight of the NPs, 2 mL of the formulation was lyophilized in glass vials, which were weighed before adding the formulation and after freeze-drying to assess the total solid mass (glass vials + formulation). 2.2.6. Diafiltration Studies Using the Nanocarriers Containing HALMD. Diafiltration is a versatile method to determine the interaction values and the nature of association of hydrophilic low molecular-weight species to excipients and formulations dispersed in aqueous medium, following a procedure widely described.66−75 Theoretically, the AE in solution may be taken comprising thermodynamically bound (TB) and kinetically bound (KB) fraction of molecules. The total fraction of drug bound, AE, independently of the mechanism of binding, is denoted by AE = TB + KB

3. RESULTS AND DISCUSSION 3.1. Chemical Interactions between Drugs and PSS To Form Nanocarriers. 1H NMR is a powerful technique that allows identifying interactions between components that produce changes in the chemical environment surrounding the protons. In particular, aromatic−aromatic interactions are easily detected because the hydration shell of the reactants changes, and magnetic fields produced by the electronic currents of aromatic groups affect the chemical shift of protons of neighbor molecules.76−78 In the 1H NMR experiments shown in Figure 2A−E, we can see the behavior of the protons present in the structures of the aromatic drug AMT in the absence of polymers and in the presence of the aromatic polymer PSS and the aliphatic polymer PVS. Spectra corresponding to IMI and CBZ are shown in the Supporting Information (see Figures S1 and S2, respectively), showing a similar behavior.

(1)

For determination of the fraction of AMT kinetically or thermodynamically bound to the nanocarriers, diafiltration studies were done. For this aim, diafiltration has been modeled as a two-compartment system, where a continuous liquid supply from the donor chamber (reservoir) is kept, maintaining a constant volume in the diafiltration cell. The unit used for diafiltration analyses consisted of a diafiltration cell (10 mL, Amicon 8010), a regenerated cellulose membrane (cutoff of 5000 Da, Merck, Germany), a reservoir, a selector, and a pressure source (Merck Millipore, Germany). For the diafiltration experiments, aliquots of 10 mL of the formulations were added into the diafiltration cell and then filtered under 3 bar of pressure and magnetic stirring. The volume in the filtration cell was kept constant during the experiment, by creating a continuous flux of liquid through the diafiltration cell, from the reservoir to the collector tube. Milli-Q water (pH 7) was used as solvent. A total of 8 samples (approx. 5 mL) were collected and AMT concentration in each sample was determined by spectrophotometry. Subsequently, the TB and KB fractions were determined as follows TB = v(km − j)/km

(2)

KB = u − um

(3)

The parameters v and u represent the initial fraction of AMT thermodynamically bound to the particles, thus in equilibrium, and the fraction bound to the nanocarriers whose release is kinetically controlled, respectively. The parameter j is related to the strength of interaction corresponding to the reversibly bound drug fraction (v). The parameters um and km correspond to u and j values, respectively, obtained in blank experiments as control performed by diafiltration of the drug in the absence of other nanocarrier excipients. Details of diafiltration procedures and results analysis are widely explained in previous

Figure 2. 1H NMR spectra (500 MHz) in D2O at pH 7 of the aromatic region of samples containing: AMT 100 × 10−3 M in the absence of any polymer (A), AMT 1 × 10−3 M in the absence of any polymer (B), PSS 1 × 10−2 M (C), AMT 1 × 10−3 M in the presence of PSS 1 × 10−2 M (D), and AMT 1 × 10−3 M in the presence of PVS 1 × 10−2 M (E). D

DOI: 10.1021/acs.molpharmaceut.9b00097 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Figure 3. Optical images of the AMT/PVS (A) and AMT/PSS (B). Turbidimetric measurements of drugs/PSS (◆) and drugs/PVS (■) formulations (C). Apparent hydrodynamic diameter (bars) and zeta potential (lines) for drugs/PSS formulations (D) [mean ± standard deviation (SD); n = 3]. Black color represents IMI/polymer formulations, orange color represents AMT/polymer formulations, and blue color represents CBZ/polymer formulations.

drugs and polymers, it was evidenced a different optical behavior, as seen by vis-spectrophotometry (Figure 3). When comparing formulations containing PVS with those containing PSS, absence of turbidity was observed in the former, while presence of turbidity in the latter (Figure 3A,B). For PSScontaining formulations, it can be inferred three tendencies evidenced by spectrophotometry as a function of the charge ratio (Figure 3C): a first zone “absence of turbidity” attributable to the presence of soluble complexes; a second zone “turbidity and absence of precipitates” which could obey to the presence of colloidal structures; and, finally, a third zone “turbidity and presence of precipitates” which is attributable to the destabilization of the colloidal behavior. Dealing with molecules undergoing electrostatic interactions, turbidity may be found when the molecules are of high molecular-weight or part of larger structures.86,87 On the contrary, ionic attractions between oppositely charged low molecular-weight drugs and polyelectrolytes do not normally produce turbidity when only primary long-range electrostatic interactions are dominant because, as explained above, the molecules conserve their hydration sphere.70,88,89 These kinds of interactions are not adequate for the formation of drug delivery systems due to the fact that these interactions are susceptible to the presence of salts and changes in the pH and also are difficult to handle.71,74 The presence of turbidity between aromatic polyelectrolytes and aromatic low molecularweight drugs witnesses the formation of nanostructures due to the occurrence of additional short-range interactions that strengthen molecular assembling, a fact of great interest for pharmaceutical delivery formulations. Thus, ion pairs may be formed due to the short-range character of the interaction, and these ion pairs aggregate conforming hydrophobic domains that lead to coacervation. In order to further characterize the formulations, all samples were submitted to DLS. As it can be seen in Figure 3D, NP formation occurs from ratios (drug/PSS) 0.55−0.75 (120−170 nm), showing negative zeta potential (−60 to −80 mV). It is interesting to underline the high zeta potential, which ensures high stability of the formulations, and the low polydispersity of the particles concerning their size distribution [polydispersity index (PDI) of 0.14−0.19, data not shown]. Interestingly, systems comprising CBZ/PSS are obtained within a narrower range (n+/n− = 0.55−0.60) compared with AMT/PSS and IMI/PSS (n+/n− = 0.60−0.75). It could be explained because CBZ has higher structural planarity (compared to IMI and

AMT is a molecule that undergoes self-aggregation at concentrations above a critical aggregation concentration (cac). The literature reports cac for this drug of around 30 mM.79−83 Our own experiments corroborate these findings, and, as can be seen in Figure S3 (Supporting Information section), a cac between 30 and 50 mM is found. The spectra of AMT at a concentration of 100 mM (self-aggregated) and at around the working concentration in this work (1 mM, free in solution) can be observed in Figure 2A,B, respectively. Although it is beyond the scope of this paper to fully characterize the geometry of the aggregate, the difference in chemical shifts is evident comparing the signals of the free molecule and of the self-aggregated drug. In the presence of PVS, we can see that, although long-range electrostatic interactions between PVS and AMT necessarily take place, they do not significantly alter the observed chemical environment of AMT protons, indicating that the molecules conserve their hydration sphere. On the contrary, in the presence of PSS, it is evident that the aromatic groups of PSS are inducing a strong upfield shifting of the AMT aromatic signals. Both aggregation of the drug in the polymer environment and specific aromatic−aromatic interactions, which imply releasing water from the hydration shell of both aromatic components to undergo close contacts, may contribute to the outstanding chemical shifting. The presence of short-range secondary aromatic−aromatic interactions between PSS and AMT has been further corroborated by nuclear Overhauser enhancement spectroscopy experiments66,67,84,85 that show cross peaks between the protons of both molecules (Figure S4, Supporting Information section). These results support the occurrence of short-range interactions between both components that potentially stabilize the formulations. 3.2. Physicochemical Characterization of Nanocarriers Containing HALMD. The drug/polymer formulations were synthesized, as described in the Experimental Section, by simply mixing two aqueous solutions at room temperature. Variables of the mixtures were explored as a function of the charge ratio between the cationic drugs IMI, AMT, and CBZ with the aromatic polyanionic polymer PSS or the aliphatic polyanionic polymer PVS (used as a negative control). We presume that the different chemical composition of the polymers (presence or absence of aromatic rings), which induces different interaction patterns, could significantly affect the respective results. After mixing the solutions comprising E


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evaluated ranges of salt concentration, pH, and temperature are beyond those found in the human body; thus, the stability of the nanocarriers is ensured. In addition, our formulations are also stable to freeze-drying and reconstitution processes in water (Figure 6D). This method is frequently used to preserve the properties of NP suspensions during storage over extended periods of time.86 This strategy, due to the total elimination of water, prevents the contamination by microorganisms while facilitates transportation due to the lower weight of the final product. Overall, the results indicate that optimal resuspension of the dried product is achieved without altering the hydrodynamic diameter and zeta potential of the original (fresh) formulations. In order to study the relation between the concentration of the components and the yield in terms of the number of NPs, the method of NP tracking analysis (NTA) was selected.92 In Figure 7, it can be appreciated that the number of formed NPs stood in the range of 2.6 × 108 and 15.1 × 108 NPs/mL. Interestingly, as also evidenced in Figure 7, there exists a direct relation between the concentration of the NPs and the turbidity observed by spectrophotometry. The above information is important because, in the absence of NTA equipment, turbidimetry could also be used to obtain the relative concentration of the NPs. 3.3. Kinetics of Formation, AE, and Drug Loading in the Nanocarriers Containing HALMD. The elucidation of the formation mechanism could be critical in order to understand the systems and projecting new formulations and uses. The stopped-flow strategy is a methodology that allows studying fast chemical reactions and is usually selected to analyze the chemical kinetics of reactions occurring not faster than several tenths of milliseconds. Plots describing the instantaneous turbidity (every 10 ms) obtained after mixing AMT and PSS in water are shown in Figure 8. The kinetic constants, calculated following a saturation equation, are K = (2.3 ± 0.1) × 10−1 s−1 for AMT/PSS 0.6, K = (2.8 ± 0.4) × 10−1 s−1 for AMT/PSS 0.65, K = (1.6 ± 0.1) × 10−1 s−1 for AMT/PSS 0.7, and K = (1.5 ± 0.2) × 10−1 s−1 for AMT/PSS 0.75. These results suggest that the kinetics of formation of the studied NPs would respond to a complex aggregation process as the drug content increases, probably responding to reordering stages. Other results considering polymeric rearrangement in complex systems in which aromatic− aromatic interactions play a role are found to present kinetic constants also in the order of 10−2 to 100 s−1.93 In an interesting work, Liu et al. could determine the kinetics of complexation of the polyelectrolytes poly (diallyldimethylammonium chloride) and poly(acrylic acid) at different charge ratios (n−/n+) by stopped flow. They also observed molecular reorganization processes occurring in the range of 10−2 to 100 s producing soluble polyelectrolyte complexes, followed by a coacervation step with kinetics in the order of seconds to days. The slower rate to generate the coacervates is related to the polymeric nature of both interacting species.94 In Table 1, the AE, drug loading, and release nature (kinetically or thermodynamically governed) studied by diafiltration are shown. Importantly, the AE is, in all cases, higher than 90%. Thus, it can be considered that the binding of the drug to the polymer is quantitative. In addition, it is worth to highlight that the drug loading fits in the range of 34−47%. These drug loading values have never been evidenced for other systems comprising HALMD (i.e., hydrogels, microgels, polymeric NPs, nanoemulsions, nanocapsules, liposomes,

AMT), which would favor the interaction between the drug and the aromatic ring in the polymer and would generate both NP formation and collapse of the system at lower drug/ polymer ratio. It is also evidenced in Figure 3D that, as the drug concentration increases, the size of the NP also tends to increase, which presumably obeys to a higher inclusion of the drug into the NP. It is interesting that the synthesis of the NPs shown here does not need any purification step (after or before elaboration). Concerning the size results, in the range of 120− 170 nm, the molecular weight of the polymer could presumably affect the extent of the molecular interaction, assembling, and rearrangement, if the molecular weight of the polymer drops too low (jeopardizing NP formation at the same reactants concentration), or if it is very high (with risk of macroprecipitation).90,91 After evidencing the formation of NPs with the three drugs (IMI, AMT, and CBZ), we decided to proceed deeper analysis by selecting AMT as model drug. As seen in electronic microscopy images (Figure 4), the NPs show spheroidal shape

Figure 4. STEM images of AMT/PSS (n+/n− 0.7) 130 000× (a), 240 000× (b).

and similar size to that obtained by DLS. The stability of the NPs has been also analyzed. As can be seen in Figure 5, the

Figure 5. Stability study of AMT/PSS NPs observed through the hydrodynamic diameter (bars) and zeta potential (lines) as a function of time. Blue color represents AMT/PSS 0.60, orange color represents AMT/PSS 0.65, black color represents AMT/PSS 0.70, and green color represents AMT/PSS 0.75. (Mean ± SD; n = 3).

hydrodynamic diameter and the zeta potential of the formulations remained practically unchanged at least for 18 days. Similarly, stable size and zeta potential were found in the presence of different salt concentration (Figure 6A), pH variations (Figure 6B), and temperature variations (Figure 6C). It is important to note that the limits of the selected F


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Figure 6. Stability study of AMT/PSS NPs observed through the hydrodynamic diameter (bars) and zeta potential (lines) as a function of different NaCl concentration (A), pH (B), and temperature (C). Hydrodynamic diameter and zeta potential variations for reconstituted freeze-dried AMT/ PSS NPs with different concentrations of trehalose (5 and 10%) (D). Blue color represents AMT/PSS 0.60, orange color represents AMT/PSS 0.65, black color represents AMT/PSS 0.70, and green color represents AMT/PSS 0.75. (Mean ± SD; n = 3).

Figure 7. NP concentration (bars) and turbidimetry (lines) of AMT/ PSS formulations (n+/n− 0.1−1.0) (mean ± SD, n = 3).

Figure 8. Kinetic curves formation of the AMT/PSS formulations (mean ± SD, n = 3).

etc.).14−16,75 On the other hand, it can be also seen that the release of AMT is mainly kinetically controlled, as more than 80% of the drug molecules are not subjected to equilibrium with the bulk.73 This fact is in agreement with Figure S5 (see Supporting Information section) showing a slow and sustained release of AMT that does not exceed 16.5% in 20 days when assayed by dialysis (pH = 7 and 37 °C). This result suggests that the fraction of AMT released corresponds to the AMT thermodynamically bound to the nanocarrier (5.9−8.8%, determined by diafiltration). Considering that the drug is a structural component of the nanocarrier, this release would provide a concomitant detachment of the polymer, exposing the subjacent material (previously subjected to kinetic interactions) to the external environment and presumably

Table 1. Values of AE %, Drug Loading (%), and Drug Associated Subjected to Kinetic (KB %) or Thermodynamic Control (TB %) for the AMT/PSS Formulations (Mean ± SD, n = 3) AMT/PSS 0.6 0.65 0.7 0.75

AE (%) 99.8 92.5 90.4 98.2

± ± ± ±

0.2 0.1 5.5 0.3

loading (%) 34.2 35.6 40.2 46.8

± ± ± ±

1.9 3.3 1.4 0.9

KB (%) 93.6 84.6 84.5 89.4

± ± ± ±

5.9 0.1 9.5 0.2

TB (%) 6.2 7.8 5.9 8.8

± ± ± ±

5.7 0.3 5.0 0.1

promoting a prolonged drug release by thermodynamic equilibrium. G


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Molecular Pharmaceutics 3.4. Final Remarks. To the best of our knowledge, this is the first article describing the efficient formation of stable nanocarriers comprising hydrophilic low molecular-weight drugs and hydrophilic polymers in which the drug, besides a functional molecule to be carried by the nanocarrier, constitutes a key structural component of the NP. This innovative approach allows an outstandingly high drug loading and slow release profiles. The dual function of the drug in these systems arises from the occurrence of short-range aromatic−aromatic interactions, producing ion pairs, with release of water from the respective hydration shells of both interacting components, that tend to aggregate and migrate to the inner polymeric hydrophobic environment, allowing the collapse of the system in the form of NPs. This also explains the need of molecular rearrangement for the formation of the NPs and that a large number of drug molecules are placed in the inner part of the NP without undergoing fast equilibrium with the bulk, thus being subjected to kinetically controlled release. In addition, the NPs, formed with hydrophilic aromatic molecules, are synthesized by simply mixing aqueous solutions at room temperature. Considering that a variety of drugs are hydrophilic, low molecular-weight molecules showing ionizable groups and aromatic rings (antihistaminic, antibiotics, antitumoral, antiarrhythmic, antidepressants, antidiabetic, antipsychotic, antibacterial, antihypertensive, nonsteroidal anti-inflammatory, and others),36,95−99 and in view of the difficulty to retain HALMD in formulations with therapeutic potential in nanocarriers,14−16,76 these results are highly encouraging for the design of a large number of new pharmaceutical formulations in the form of nanocarriers comprising drugs against different pathologies.

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AUTHOR INFORMATION

Corresponding Authors

*Correspondence to: E-mail: [email protected], Phone: +56632293520 (I.M.-V.). *E-mail: [email protected], Phone: +56229781616 (F.A.O.-A.). ORCID

Toshimichi Shibue: 0000-0003-4766-1896 Hiroyuki Nishide: 0000-0002-4036-4840 Ignacio Moreno-Villoslada: 0000-0003-4125-1220 Felipe A. Oyarzun-Ampuero: 0000-0002-4951-0701 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by FONDECYT 1161450 (F.A.OA.), FONDEQUIP EQM160157 (F.A.O-A.), FONDEQUIP EQM170111 (F.A.O-A.), FONDEQUIP EQM-150106 (J.G.), FONDECYT 1181695 (I.M.-V.), and CONICYT-FONDAP 15130011 (F.A.O-A).

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REFERENCES

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4. CONCLUSIONS In this work, a new, green, and highly efficient methodology to noncovalently attach HALMD to hydrophilic aromatic polymers to create nanocarriers is presented. The proposed method is simple and consists in mixing an aqueous solution containing HALMD with another aqueous solution containing the aromatic polymer. NMR experiments demonstrates strong chemical shifting of HALMD aromatic protons when interacting with PSS, evidencing aromatic−aromatic interactions. The obtained nanocarriers are spheroidal, showing a size range of 120−170 nm, low polydispersity (PDI ≤ 0.2), and negative zeta potential (from −60 to −80 mV). Importantly, in addition to high drug AEs (≥90%), the formed nanocarriers show drug loading values never evidenced for other systems comprising HALMD reaching ≈50%. Diafiltration and stopped flow experiments evidenced a kinetic drug entrapment governed by molecular rearrangements. Considering the large quantity of HALMD with importance in therapeutics, and the simplicity of the presented strategy, we envisage these results as the basis to elaborate a number of drug delivery systems with applications in different pathologies.

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presence of PSS and PVS; aggregation assay of AMT; NOESY experiment of AMT/PSS formulation; in vitro release of AMT from AMT/PSS formulations versus free AMT; and results of the diafiltration experiments (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.9b00097. 1 H NMR spectra (500 MHz) in the aromatic region of IMI and CBZ in the absence of any polymer and in the

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