Polymersomes Based on a Fumaric Copolymer as a Novel Drug Nanocarrier

doi:10.21203/rs.3.rs-3403580/v1

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Polymersomes Based on a Fumaric Copolymer as a Novel Drug Nanocarrier

https://doi.org/10.21203/rs.3.rs-3403580/v1

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There is great interest in the development of nanoparticles as new platforms for the transport and release of drugs that present drawbacks for their direct administration into the body. In this work we developed a platform for bisphosphonates encapsulation based on polymersomes. Polymersomes were obtained by self-assembly of a new triblock amphiphilic copolymer based on polyethylene glycol (PEG) as hydrophilic block, and a statistical copolymer of vinyl benzoate (VB) and diisopropyl fumarate (DIPF) as hydrophobic block. The copolymer was synthesized by reversible addition fragmentation chain transfer polymerization (RAFT). The obtained amphiphilic polymer presented a weight fraction of hydrophilic block (f) of 30, appropriate for the formation of vesicles by self-assembly. Using the solvent injection method, we obtained vesicles of around 54 nm with a Z-average hydrodynamic diameter of 163 nm. The nanoparticles were smaller and with narrow size distribution than particles formed by the counterpart copolymer without DIPF. In order to evaluate the potential of these copolymers as a vehicle for bisphosphonates, risedronate loaded polymersomes were prepared. The obtained particles showed a similar size to their uncharged analogues, with a content of 12 ± 2 mg of risedronate per g of polymer, determined by HPLC. Cell viability and cytotoxicity were evaluated using murine monocyte-macrophage RAW264.7 cells. The empty or risedronate-loaded nanoparticles did not affect cell proliferation, viability and nitric oxide production at the tested concentrations.

Polymersomes

Nanocarrier

Bisphosphonates

Fumaric copolymer

Triblock copolymer

To overcome the problems of direct administration of drugs into the body, various nanosystems with different compositions and biological properties have been extensively investigated. These systems could be used to improve the uptake of poorly soluble drugs that in turn could improve bioavailability of drugs [1, 2]. Thus, drug delivery modalities based on nanoparticles (NPs) have been intensively investigated for their use in diverse therapies [3, 4]. Among the different NPs, organic NPs like liposomes, micelles, dendrimers, and polymeric NPs (nanospheres and nanocapsules) have been intensively investigated and are the mostly used as drug delivery systems [5]. The most common forms of polymeric NPs are nanospheres (solid matrix systems) and nanocapsules: cavities surrounded by a polymeric membrane or shell, like polymersomes, micelles and dendrimers.

Polymersomes are artificial vesicles, with membranes made using amphiphilic block copolymers which can self-organize into this nanostructure when the fraction of hydrophilic block (f) is, as a general rule, around 35 [6], but it is also dependent of other parameters such as the chemical composition and dispersity of the polymer blocks, temperature, and concentration of polymer [7]. Polymersomes are comparable to liposomes, although the first are reported to have improved stability and cargo-retention efficiency [8].

The advances in novel-controlled polymerization techniques have allowed the design of a wide variety of amphiphilic block copolymers which can self-organize into polymersomes. While diblock copolymers are intensely used in the development of polymersomes [9, 10], the use of triblock copolymers is less frequently reported [11, 12]. When biomedical applications are involved, the selection of the materials responds not only to the hydrophilic-hydrophobic nature of the components, but also to the biocompatibility requirements. Until yet, an interesting material which has not been included in these nanosystems are the fumaric monomers. Homo- and copolymers of fumaric esters have been studied in our group for some years, not only to clarify the polymer structure-property relationship but also for glimpse insight of the potential applications that can be derived from them [13–15]. Considering their properties, fumaric polymers can be considered competitive with poly(methacrylic esters), besides that fumaric ester precursors could be produced by fermentation processes from renewable resources [16, 17]. Indeed, polydialkyl fumarates are probed biocompatible materials [18, 19], which became useful for drug delivery applications.

Bisphosphonates are first line drugs osteoporosis treatment [20, 21]. Risedronate, nitrogen containing bisphosphonate, exhibits low oral bioavailability and it causes gastrointestinal irritation as main adverse effect which compromises compliance [22–24]. On the other hand, risedronate has a very short half-life (1.5h) and requires frequent oral administration [25]. Different strategies have been proposed as alternative to the actual administrations of bisphosphonates [26], for instance microparticles, nanodispersions or transdermal drug delivery system [25, 27, 28]. Regarding to the nanoparticle system, risedronate-hydroxyapatite nanoparticles [29] cationic liposomes and polylactide-co-glycolic-acid microspheres [30, 31] and lipid-based systems have been proposed [32]. Polymersomes has not been extensively studied as an encapsulation and delivery platform for oral bisphosphonates administration.

We have previously reported the development of biocompatible polymersomes by self-assembly of amphiphilic triblock copolymers. These nanoparticles were based on polyetilenglicol (PEG) and poly(vinyl benzoate) (PVB) as hydrophilic and hydrophobic block, respectively [33]. The particles showed suitable dimensions and morphology to be used as potential nanocarriers of vitamin D3 [34].

The aim of the present work was the development of polymersomes based on PEG and fumaric copolymer as risedronate delivery system. In this study, a new amphiphilic triblock copolymer composed by PEG and a fumaric copolymer were synthesized by reversible addition fragmentation chain transfer polymerization (RAFT), and used for the preparation and characterization of polymersomes. Since the biocompatibility of bulk biomaterials cannot be extrapolated to these particles, we evaluated cell viability and toxicity of the obtained polymersomes. Risedronate loaded nanovesicles were also obtained to evaluate the capability of the system as nanocarrier of this bisphosphonate.

Materials

Diisopropyl fumarate (DIPF) monomer is not a commercial product, was prepared and purified as previously described [13]. Briefly, a mixture of fumaric acid and isopropanol (ratio 1:2.65) were added to toluene, in the presence of catalytic amount of sulfuric acid which was kept under reflux during 22 h. The product was purified by distillation under vacuum (yield 76%). Risedronate [2-(3-pyridyl)-1-hydroxy-ethane-1,1-bisphosphonate monosodium salt] was obtained of a sample synthesized previously in our laboratory [27]. Dimethyl 2,2′-azobis(iosbutyrate) (MAIB) was synthesized in our laboratory from 2,2´-azobis(isobutironitrile) (AIBN), following the protocol reported in the literature [35, 36]. PEG6000 (≥ 99%, BiopaCK), Vinyl Benzoate (VBz, 99%, Sigma-Aldrich), Benzoyl peroxide (BP, Merck), Tetrahydrofurane (THF, Cicarelli), Toluene (Anedra) Methanol (MeOH, Merck, for liquid chromatography), Tetrabutylammonium bromide (TBAB, Merck), KH₂PO₄ (Anedra), Na₂HPO₄ (Merck), Phosphoric acid (Anedra) were the analytical grade a used as received.

Synthesis of block copolymers

The PVBz-b-PEG-b-PVBz (S1) and P(VBz-co-DIPF)-b-PEG-b-P(VBz-co-DIPF) (S2) triblock copolymers were synthetized by reversible addition-fragmentation chain transfer (RAFT) polymerization using a dixanthate terminated PEG as macro-chain transfer agent (PEG macro-CTA), based on the procedures previously described [33]. In the case of S2 synthesis, the procedure was adapted for the inclusion of the DIPF monomer in the reaction mixture using dimethyl 2,2′-azobis(iosbutyrate) (MAIB) as initiator (PEGX macro-CTA (3.58 mM), vinyl benzoate (1.81 mM), DIPF (1.80 mM) and MAIB (1.84 mM) in toluene (60 wt%). The resulting copolymers included poly(ethylene glycol) (PEG) as hydrophilic block, and poly(vinyl benzoate) (PVBz), or poly(vinyl benzoate-co-diisopropyl fumarate) (P(VBz-co-DIPF) as hydrophobic block.

Self-assembly of block copolymers

Preparation of polymersomes was carried out by self-assembly of synthesized triblock copolymer as previously indicated [33]. Briefly, ten milligrams of copolymer were dissolved in 1 mL of THF and 10 mL of Milli-Q water was added slowly with a flow rate of 30 ml/h under magnetic stirring (600 rpm) at room temperature.

Methods

¹H NMR spectrum of copolymer was recorded with a Bruker-500 MHz (Avance-AV 500) in deuterated chloroform (DCl₃) at 25°C. Tetramethylsilane (TMS) was used as an internal standard.

A Shimadzu FTIR (IRAffinity-1) spectrometer was used to record the infrared spectra of copolymers as a thin film onto a sodium chloride (NaCl) window, between 4000 and 400 cm^− 1 with a resolution of 4 cm^− 1 and 32 accumulated scans. Spectrum analysis was performed using EZOMNIC software (EZOMNIC 7.4.127, Thermo Fisher Scientific Inc, Madison, WI, USA).

The average molecular weights (M_n and M_w) and polydispersity index (PDI) were determined by size exclusion chromatography (SEC) with an LKB-2249 instrument at 25°C. A series of four µ-Styragel columns, ranging in pore size 10⁵, 10⁴, 10³, 100 Å were used with chloroform as eluent. The sample concentration was 4–5 mg/ml and the flow rate was 0.5 ml/min. The polymer was detected by UV detector (Shimadzu SPD-10A) at a wavelength of 254 nm. Calibration was done with polystyrene standards supplied by Polymer Laboratories and Polysciences. The fraction of hydrophilic block (f) was estimated from the PEGX and triblock copolymer M_n relationship.

Transmission electron microscopy (TEM) of polymersomes was made with a JEOL 1200 EX II microscope and Phillips CM200-UT LaB6), both operated in bright field condition. The sample was adsorbed onto formvar-coated copper grids and negatively stained with 0.5% (wt/v) phosphotungstic acid and the pH adjusted to 7. Images were analyzed using Image J software (www.macbiophotonics.ca/imagej) with a calibration plugin to determine the particle-mean diameter. Results are expressed as the average ± SEM.

Dynamic light scattering (DLS) (Nano ZS Zetasizer-ZEN3600, Malvern Instruments Corp., UK) was performed in order to analyze the hydrodynamic mean diameter, size distribution, and polydispersity index (PDI). The equipment includes a 4-mW He–Ne (633 nm) laser and the measurements were carried out in water at 25°C. Data was analyzed using CONTIN algorithms (Malvern Instruments). Diluted solutions (0.25 mg/ml) of the polymersomes with or without risedronate were used to carry out the measurements and the results were expressed as a six-measurement average.

In vitro biocompatibility assay

Murine monocyte-macrophage RAW264.7 cells were cultured in Dulbecco’s Eagle modified media (DMEM, Invitrogen Argentina) supplemented with 10% fetal bovine serum (FBS, Internegocios S.A., Argentina) in a humidified atmosphere of 95% air and 5% CO₂.

Cell proliferation was evaluated using the crystal violet bioassay, routinely performed in our laboratory. Briefly, cells were incubated with increasing concentration of polymersomes during different periods of time in either DEMEM-10% FBS or DEMEM. At the end of the assay, cell monolayer was washed and fixed with 5% glutharaldehyde/PBS, stained with 5% crystal violet in methanol and color was extracted with 0.1% glycine/methanol buffer. The resulting absorbance was read at 540 nm.

For cytotoxicity assays cells were cultured on multiwell culture dishes in DMEM with or without 1% FBS, and incubated during 24, 48 or 72h with increasing volumes of polymersomes (1, 5 and 10 µL of 1.6 µg/mL polymersomes dispersion). At the end of the experiment, cells were incubated for an additional hour with the tetrazolium salt MTT to produce formazan. After washing, the formazan precipitate was dissolved in dimethyl sulfoxide (DMSO) and the absorbance read at 570 nm.

Additionally, basal nitric oxide (NO) production was evaluated by the Griess’ method. Briefly, cells were cultured with or without polymersomes during different periods of time in DMEM without phenol red. Then 500 µl of conditioned media was collected and added to 500 µl of reactive Griess’ solution as previously described [37]. Absorbance was read at 550 nm. Cell monolayer was lysed with 200 µl 0.1% Triton X100 and total protein content determined by Bradford’s method.

Drug load evaluation

Polymersomes loaded with risedronate were obtained by using the passive loading strategy by adding the drug to the aqueous phase (1 mg/ml) in which the vesicles form, following the procedure described above (section 2.3). After loaded polymersomes formation, samples were dialyzed to remove the risedronate not included in the vesicles (dialysis tubing cellulose membrane, cut-off 14000, Merck). The obtained dispersions were analysed by TEM and DLS. Evaluation of drug encapsulation efficiency was performed in lyophilized water dispersion of risedronate-loaded polymersomes. Briefly, 10 mg of the solid obtained after lyophilization was dissolved in CHCl₃ (0.50 ml) and then solvent extraction of risedronate was performed adding 1.00 ml of buffer (5 mM tetrabutylammonium bromide (TBAB), 2.5 mM KH₂PO₄, 8.5 mM Na₂HPO₄, pH 7.45). The obtained water phase containing the risedronate was analysed by reverse-phase high-performance liquid chromatography (RP-HPLC) for the determination of the load of the vesicles. Hewlett Packard HP 1100 HPLC equipment with UV detection was used (diode array detector set at 262 nm). Chromatographic separations and subsequent quantifications were carried out at 25°C using a ZOBAX Eclipse XDB-C18 (4.6 × 150 mm, 5 µm) column. The mobile phase consisted of buffer (5 mM TBAB, 2.5 mM KH₂PO₄, 8.5 mM Na₂HPO₄, pH 7.45) − methanol (75:25 (v/v)), and was filtered before use (Nylon membranes, 0.45 µm,13 mm). The flow rate was adjusted to 1.2 mL/min at 25◦C. The injection volume was 20 µL and each sample, previously filtered through 0.45 µm filters, was analysed in triplicate. Peak areas were used for quantitative analyses. Calibration curve of peak areas versus risedronate concentration was made with different dilutions of a stock solution (1 mg/mL), which was linear between 1 µg/mL and 50 µg/mL. Retention times (t_R) and peak areas were evaluated using ChemStation Software.

Statistical analysis

Bonferroni multicomparison test was used for comparisons between the control and experimental groups. All results are expressed as mean ± SEM and represent at least three different experiments performed in triplicate.

Synthesis of PEG macro-CTA and triblock copolymers

PEG macro-CTA was synthesized according to the reaction procedure described previously and characterized by spectroscopic measurements [33]. It showed an increase in the UV absorbance at 280 nm confirming the inclusion of the xanthate group on the PEG structure, and presented the typical FTIR and ¹H-NMR signals described in the literature. PEG macro-CTA: Yield 38%. FTIR (ν, cm^− 1) 2948, 2877 (C–H), 1734 (> C = O), 1114 (C–O), 1060 (> C = S), 843 (> C–S). ¹H-NMR (CDCl₃): (δ, ppm), 4.63 (CH₃–CH₂–OC(O)), 4.40 (> CH–CH₃), 4.23 (–CH₂–OC(S)), 3.60–3.85 (–CH₂–CH₂–O), 1.60 (> CH–CH₃), 1.30 (CH₃–CH₂–OC(O)). UV(> C = S), CHCl₃: (π→π*, λ_max = 280 nm) and (n→π*, λ_max = 357 nm).

Block copolymers were synthetized from PEG macro-CTA. Both samples were characterized by FTIR and ¹H-NMR. In S1 polymer, typical signals of the PVBz and PEG blocks were observed in agreement with our results previously reported (data not shown) [33]. The new copolymer S2 showed the FTIR bands located at 3062 cm^− 1 (νC-H aromatic), 2921 cm^− 1 and 2849 cm^− 1 (νC-H aliphatic), 1723 cm^− 1 (νC = O), and 1271 and 1115 cm^− 1 (νCO–OR). ¹H-NMR spectrum of this sample is shown in Fig. 1, including the structure and the peaks assignation based on the corresponding homopolymers spectra, which confirms the structure of the obtained polymer.

The degree of polymerization (DP), composition and ratio of the hydrophilic to hydrophobic blocks, are critical in order to produce vesicles by self-assembly. It is important to define and control these parameters to deepen in the relationship polymer structure/vesicle morphology knowledge to the purpose to obtain the desired vesicle morphology [38].

Based on the ¹H-RMN results, composition of copolymer was estimated from the integral ratio of the peak at 3.6 ppm corresponding to methylene hydrogen of ethylene glycol (EG) unit, the signal at 7.7 ppm corresponding to the hydrogen of the aromatic ring of VB unit, and the signal at 4.9 ppm corresponding to the methyne hydrogen of DIPF unit, using the ratio of Eq. (1) for the S2 copolymer:

$${F}_{PEG}=\frac{{I}_{1}/4}{{I}_{1}/4+ {I}_{2}/2+{I}_{3}/1}$$

where F_PEG is the molar fraction of EG unit in the copolymer and I₁, I₂ and I₃ represent the ¹H-NMR resonance peak areas at 3.6 ppm, 7.7 ppm and 4.9 ppm, respectively. Similarly, Fs of the other monomers in the block copolymers were calculated. The relative composition of the EG:BV:DIPF was 0.36:0.28:0.36. Thus, this copolymer presented a hydrophobic block with a relative composition of 44:56 (percentage mole fraction) of BV: DIPF, respectively.

Figure 2 shows the SEC results for the PEG macro-CTA and the triblock copolymers synthesized in this work and Table 1 summarizes the percent of conversion (C), number average molecular weight (Mn), polydispersity index (PDI), and fraction of hydrophilic block of synthetized copolymers. Macro-CTA presented a number-average molecular weight (Mn) of 8806 g/mol and a polydispersity index (PDI = Mw/Mn) of 1.1. After growth of hydrophobics blocks, an increase of the molecular weight and PDI were observed. The copolymer PVBz-b-PEG-b-PVBz showed a Mn of 22.00 kg/mol, PDI of 3.40, and f = 38. These parameters for P(VBz-co-DIPF)-b-PEG-b-P(VBz-co-DIPF) were: Mn = 31.50 kg/mol, PDI = 1.95 and f = 30. These results evidence the increase in molecular weight as a consequence of the hydrophobic blocks growth. In addition, the chain length obtained produce an f adequate for the formation of vesicles by self-assembly.

Table 1

Characteristics parameters of the synthesized copolymers.
Sample name	Composition	time [h]	C [%]	M_n [Kg/mol]	PDI	F^a [%]
Macro-CTA	PEG-xanthtate	-	38	8.8	1.10	-
S1	PVBz-b-PEG-b-PVBz	16	41	22	3.4	38
S2	P(VBz-co-DIPF)-b-PEG-b-P(VBz-co-DIPF)	17	39	31.5	1.95	30
^a Based on SEC results Percent of conversion (C), number average molecular weights (Mn), and polydispersity index (PDI).

Self-assembly of the triblock copolymers

It has long been recognized that polymersome or polymeric vesicles made of self-assembled amphiphilic block copolymers are very promising nanostructures [39]. They appear to be highly stable, and present the advantage to encapsulate hydrophilic drugs in the core and hydrophobic drugs in the shell. Thus, they can act as biocompatible devices with wide application in healthcare. In this sense, polymersomes development is a field of great interest and intense research [40]. Our group is devoted to the development of delivery systems for bone pathologies and, in view of these applications the amphiphilic triblock copolymers previously described were self-assembled to generate polymersomes. We were also interested in the effect of the nature of the hydrophobic block on the nanoparticle characteristics. Figure 3 shows TEM images and the size distributions of polymersomes prepared from samples S1 and S2. We found that amphiphilic triblock copolymers formed spherical particles (Fig. 3a and b), but particles obtained from S2 (statistical fumaric copolymer as hydrophobic block) presented smaller average diameter than those obtained from S1 (PVB as hydrophobic block) (Fig. 3b and d). Furthermore, in S2 sample a broad size distribution was observed, although the content of large particles is very small.

Polymersomes obtained from S1 and S2 samples were also analyzed by dynamic light scattering (DLS) and the intensity distribution curves are presented in Fig. 4. It was observed that sample S1 shows a monomodal distribution with a hydrodynamic Z-average diameter (Z_D) of 163 ± 64 nm and polydispersity index (PDI) of 0.25 ± 0.03 which indicated a narrow size range, considering that the shoulder near 450 nm is of much lower intensity. In comparison, sample 2 exhibited a monomodal distribution with a hydrodynamic Z-average diameter (Z_D) of 119 ± 17 nm and polydispersity index (PDI) of 0.18 ± 0.01 indicating a narrower size range than sample S1.

For both samples, the size differences measured by the TEM and DLS techniques, are in agreement with those previously reported by us and other researchers [33, 34, 41, 42]. These results are consistent with previous studies indicating the size of the polymersomes is influenced by not only the molecular weight of the hydrophobic block, but its chemical nature, composition and rigidity [6]. In our work we found that particles prepared from random copolymer as hydrophobic block have smaller size than those prepared from polyvinylbenzoate as hydrophobic block, emphasizing the influence of the hydrophobic block composition in particle formation.

Characterization and entrapment efficiency of risedronate

The morphology of the vesicles from samples S1 and S2 incorporating risedronate were characterized by TEM and DLS. In Fig. 5 shows the TEM images of both samples together with the corresponding size distribution. It can be observed that sample S1 exhibit a greater average diameter than those of sample S2. Besides, both size distributions are narrow with a very small contribution from large particles. These results are similar to those observed in empty vesicles (Fig. 3).

The curves intensity vs size distribution obtained for dynamic light scattering (DLS) of both samples incorporating Risedronate (R) are presented in Fig. 6 (S1 + R and S2 + R). Both samples presented a bimodal distribution with a main peak (P1) corresponding to the one with the highest integral intensity and a second peak (P2) corresponding to a second smaller size population. The maximum value of distribution in intensity of each peak along with the PDI values are presented in Table 2. We also included the values for the samples without drug (S1 and S2) for comparison purposes.

Table 2

Maximum of the intensity distribution of each particle´s population and polydispersity index.
Sample	P1 [nm]	P2 [nm]	PDI
S1	137 ± 4	468 ± 73	0.25 ± 0.01
S1 + R	144 ± 2	519 ± 73	0.22 ± 0.01
S2	166 ± 11	63 ± 8	0.18 ± 0.01
S2 + R	179 ± 4	67 ± 5	0.18 ± 0.01

In both cases (S1 and S2), data presented seems to show a small increase in the size of the vesicles by incorporating the hydrophilic drug. However, such size increase does not correspond to statistically significant differences. This result is concordant with hydrophilic properties of the encapsulated drug, that remains included in the internal aqueous portion of the vesicles, without affecting their dimensions.

To evaluate efficiency of encapsulation (amount of active principle encapsulated in the polymersomes) we carried out RP-HPLC assays. The analysis of RP-HPLC chromatograms revealed the presence of the characteristic peak of risedronate at t_R = 1.824 ± 0.004 min. Additionally, chromatograms of polymersomes risedronate-loaded samples after extraction, presented a peak corresponding to the drug. The analysis of the area under the peak revealed that the content of risedronate in polymersomes was around 4 ± 2 and 12 ± 2 mg of risedronate per gram of polymersomes for samples S1 + R and S2 + R, respectively. In S1 + R sample, the bisphosphonate concentration for osteogenic in vitro effect was suboptimal, while in S2 + R risedronate concentration was in the optimal range to achieve the same osteogenic effect [43].

3.3 Biocompatibility assays

In living beings, macrophage cells from immune system act as important immune sentinels and are able to react against every particle sensed as foreign body [44]. To determine the in vitro biocompatibility of synthesized polymersomes, we selected an established cell line of murine macrophage/monocytes.

S2 derived–polymersomes allowed cell proliferation at same extent as control condition, without differences over time, as determined by crystal violet method (Fig. 7a). This effect of S2 polymersomes on RAW264.7 proliferation was independent of the tested concentration. In accordance, our previous cytotoxicity studies using RAW 264.7 macrophages incubated with S1 polymersomes demonstrated the absence of cytotoxicity during the same tested period of time [33].

Then, we evaluated mitochondrial activity through the MTT bioassay. This assay is based on the reduction of MTT by mitochondrial succinate dehydrogenase to form an insoluble dark blue formazan product [45]. Only viable cells with active mitochondria reduce significant amounts of MTT to formazan, indicating cell viability. We found that cell viability was preserved in all studied conditions over time (Fig. 7b).

Similar results were obtained with S2-risedronate loaded polymerosomes (S2 + R) they allowed cell proliferation at same extent as control condition, without differences over the culture time (Fig. 7c).

Next, we designed a series of experiments to evaluate a possible anti-inflammatory activity. RAW264.7 cells were incubated either in presence of polymersomes and /or bacterial lipopolysaccharide (LPS) for 24 h, and then we evaluated cell proliferation and nitric oxide production. We found that LPS treated cell proliferate less (80%, p<0.01) compared to basal condition (Fig. 8a), both in presence of 1% of FBS or in DMEM without FBS. While, nanoparticles reverted this toxic effect (nanoparticles alone had no effect on cell proliferation, Fig. 8a). In accordance, NO production was increased in the presence of LPS (42 folds vs basal, Fig. 8b, p<0.0001), while 10 µL of nanoparticles partially reverted this effect (35 folds vs basal, Fig. 8b, p<0.0001). On the other hand, nanoparticles induce a moderate increase on NO production (13 fold vs basal, Fig. 8b) without affecting cell viability or proliferation (Fig. 7). To determine if the effect on nitrite determination was an interference of polymersomes, the control nitrite curve was performed in the presence or absence of polymersomes. Figure 8c shows that there are no differences between points, suggesting that there is other mechanism than direct quenching of nitrite in culture media.

The observed absence of response using RAW264.7 macrophages was not totally surprising, since it has been previously described that those particles containing PEG residues in its structures has low protein affinity as well as induces no immune response [46]. Moreover, it has been demonstrated that liposomes obtained from PEG modified polymers are more resistant to protein interaction, plasma clearance and macrophage uptake [47]. Although additional studies are needed in order to evaluate the release profile of the drug and the effects that it can cause, our findings open a lot of opportunities in the design of therapies based on bisphosphonates-loaded polymersomes.

In the present work we have developed a nanocarrier platform based on triblock copolymers of polyethylene glycol (PEG) as hydrophilic block, and a statistical copolymer of vinyl benzoate (VB) and diisopropyl fumarate (DIPF) as hydrophobic blocks. We demonstrate that both the morphology and size of the nanoparticles depend on various factors such as the chemical structure and comonomer composition of the copolymer, as well as the relationship between hydrophobic/hydrophilic blocks. We also explored the ability of polymersomes to encapsulate bioactive hydrophilic drugs in its core and its biocompatibility properties, obtaining systems that allowed cell proliferation and viability under the conditions tested. The obtained results showed that the prepared systems are a great candidate as drug delivery of active principles related to the prevention and treatment of bone disorders.

Acknowledgements

The authors thank Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP, 11/X769) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) (PICT-2018-01931) for finantial assistance. RB is fellowship of ANPCyT. SM, JA, MSC and PJP are members of the Carrera del Investigador Científico CONICET.

Conflict of interest: The authors declare that they have no competing interests.

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Polymersomes Based on a Fumaric Copolymer as a Novel Drug Nanocarrier

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