Effect of crosslinking density on thermoresponsive nanogels: A study on the size control and the kinetics release of biomacromolecules

European Polymer Journal 124 (2020) 109478

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Effect of crosslinking density on thermoresponsive nanogels: A study on the size control and the kinetics release of biomacromolecules Lucila Navarroa,b, Loryn E. Theunea, Marcelo Calderóna,c,d,

T

⁎

a

Freie Universität Berlin, Institute of Chemistry and Biochemistry, Takustr. 3, 14195 Berlin, Germany Instituto de Desarrollo Tecnológico para la Industria Química (INTEC), Universidad Nacional del Litoral (UNL) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Colectora Ruta Nac 168, Paraje El Pozo, CP 3000 Santa Fe, Argentina c POLYMAT and Applied Chemistry Department, Faculty of Chemistry, University of the Basque Country UPV/EHU, Paseo Manuel de Lardizabal 3, 20018 Donostia-San Sebastián, Spain d IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain b

A B S T R A C T

Thermoresponsive nanogels emerged as a robust and efficient platform for the delivery of a number of therapeutic agents. The need to deliver a wide range of biomolecules from different sizes requires a structural system with the ability to be tuned according to the specific applications. In this study, we design a library of nanogels using dendritic polyglycerol (dPG) as a crosslinker and poly (N-isopropyl acrylamide) as connecting thermoresponsive chains. Nanogels with diameters in a range of 70–210 nm were obtained by precipitation polymerization with low polydispersity. We provide a detailed study on how the flexibility of the network and size of the nanogels can be tuned by the modification of functionalities of the dPG, monomer feed and the size of the crosslinker. Additionally, we use different molecular weight proteins (6–66 kDa) as an indicator of the density of the structure by encapsulation and release experiments. We prove that the versatility of the system relays on the correct design of the nanogels and the careful selection of the starting materials and its final proportion in the reaction. By controlling the network density, a wider range of application can be achieved from a system with a short-term release triggered by temperature to a long-term release that could serve as reservoir platform.

1. Introduction Nanogels (NGs) are nano-sized hydrogel particles made of crosslinked polymers [1–3]. They have the ability to swell by absorption of large amounts of water without being dissolved, which gives them the capability of holding large amounts of small molecular therapeutics, bio-macromolecules, and inorganic nanoparticles [4]. Their final properties such as size, network flexibility, ionic charge and swelling capacity can be tuned by carefully selecting their chemical composition [5,6]. Moreover, the versatility of their structure allows to introduce response towards an external or environmental stimuli such as temperature, pH, ionic force, or magnetic field, that induce a change in their physicochemical properties [7–9]. This stimuli-responsive behavior, adding to the nanoscale size, renders them with great potential for biomedical applications such as controlled drug release [7,10–15], gene delivery [16–20], biosensors [21–24], and imaging [25–27]. Poly (N-isopropyl acrylamide) or PNIPAM is a widely used thermoresponsive linear polymer that has a lower critical solution temperature (LCST) of 32–34 °C in water. When the temperature exceeds this value the polymer undergoes a reversible change in its solvation

state. During this transition, intra- and inter-chain hydrogen bonds are formed between the amide groups inducing a de-hydration and subsequential collapse of the linear polymer. This transition can be characterized and measured by the cloud -point temperature (tcp) method evaluating the transmittance of the polymer solution upon heating/ cooling [28–30] PNIPAM-based NGs are particularly interesting for drug delivery application because the collapse of the system will occur near the physiological range (32–34 °C). In contrast to linear thermoresponsive polymers, three-dimensional cross-linked NGs exhibit a temperature-dependent shrinkage or swelling behavior due to the change of the solvation state of the polymers used when the so-called volume phase transition temperature (VPTT) is exceeded. The VPTT can be determined by monitoring the size of the NGs upon heating/cooling, for example, by dynamic light scattering (DLS). In addition, due to increased density of the collapsed NGs or particles this transition is characterized by a decrease in the transmittance of the polymer solution and temperature-dependent turbidity measurements yield the so called tcp [27–29]. For PNIPAM-dPG NGs, above the VPTT the PNIPAM moieties collapse inducing the shrinkage of the NGs with the simultaneous

⁎ Corresponding author at: POLYMAT and Applied Chemistry Department, Faculty of Chemistry, University of the Basque Country UPV/EHU, Paseo Manuel de Lardizabal 3, 20018 Donostia-San Sebastián, Spain. E-mail address: [email protected] (M. Calderón).

https://doi.org/10.1016/j.eurpolymj.2020.109478 Received 5 November 2019; Received in revised form 21 December 2019; Accepted 2 January 2020 Available online 03 January 2020 0014-3057/ © 2020 Elsevier Ltd. All rights reserved.

European Polymer Journal 124 (2020) 109478

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Fig. 1. (A) Schematic representation of the cargo release triggered by temperature of dPG/PNIPAM nanogels. (B) Schematic synthesis of the nanogel synthesis. The depicted structure of dPG-Ac is a small representation of a polymer with average MW of 10 kDa.

them can diffuse out of the NGs through the pores when the NGs are in their swollen state. To overcome this issue a denser network can be designed in order to generate smaller pores but at the same time it needs to be flexible enough to swell and collapse in order to trigger the release. With the purpose of tuning the network density and flexibility, different strategies were used in this study. We evaluated the effect of the functionalization degree of the dPG, the variation of the monomer proportion on the feed and the use of two different crosslinker sizes. We performed a detailed study on how these parameters affect the thermoresponsiveness and network flexibility of the NGs. Moreover, different molecular weight proteins (6–64 kDa) were used for encapsulation and release experiments as an indicator of differences in the mesh structure of the NGs network.

expulsion of water and, if present, a therapeutic load (Fig. 1A). The crosslinking of PNIPAM with dendritic polyglycerol (dPG) has been developed by our group using precipitation polymerization method yielding well-defined NGs in a range of 100–200 nm with low polydispersity [4]. Using this strategy, dPG-NIPAM NGs are formed by radical polymerization of acrylated dPG and NIPAM at a temperature over the LCST of PNIPAM. When a certain PNIPAM chain length is reached, PNIPAM undergoes a phase transition which results in nucleation events. The growing nucleates are stabilized by a surfactant (SDS) to yield nanogels with low polydispersity (Fig. 1B) [4]. Recently, we have proven the efficacy of dPG-NIPAM NGs for cutaneous delivery using high molecular weight proteins such us bovine serum albumin (BSA), L-asparaginase II, transglutaminase, and etanercept [6,31–33]. Having a VPTT of 32 °C, dPG-PNIPAM NGs have the ability to release the cargo passing through the skin barrier which significantly enhance the penetration depth of the proteins when compared to conventional formulations [6,31]. Additionally, these NGs can accumulate in the hair follicle canals and this behavior can be exploited to create a long-term reservoir containing high drug concentrations for a sustained release to the skin [34]. The success of the NGs as penetration enhancer is based on their swelling capability to hydrate the skin and the mechanical properties that allow them to be deformed when an external force is applied (i.e. massage to promote penetration) [35]. The control of the swelling degree in an aqueous environment and the flexibility of the network are the most important properties. They can be controlled by structural features like chemical structure of the polymer matrix and crosslinking density of the NGs [2]. In this matter, the increase in the density of the network could lead to the formation of a more rigid structure, but it can also help to reduce the size of the pores of the NG, thereby enabling the retention of small molecular weight proteins. In this matter, a well-defined equilibrium of these properties must be found to assure the success of the final application. High molecular weight proteins have been successfully encapsulated with high efficiency using NGs synthesized with 10 kDa dPG and NIPAM (30/70 ratio feed) [6,31]. However, the encapsulation of small proteins is a more complex procedure due to the fact that some of

2. Materials and methods 2.1. Materials The following reagents were used as purchased: acryloyl chloride (Ac-Cl, 97%, SIGMA), potassium persulfate (KPS, 98%, SIGMA), sodium dodecyl sulphate (SDS, ≥98%, SIGMA), fluorescein 5-isothiocyanate (FITC, 97%, Merck); dry N,N-dimethyl formamide (DMF, 98%, SIGMA), triethylamine (TEA, 99%, Acros Organics). N-isopropyl acrylamide (NIPAM, 99%, Merck) was recrystallized in n-hexane before use. 2.2. Methods 2.2.1. Analytical methods 1 H NMR spectroscopy. 1H NMR spectroscopy was performed to evaluate the acrylation degree of the dPG and the PNIPAM/dPG ratio within the thermoresponsive NGs. All measurements were carried out on a Bruker DRX 400 NMR spectrometer (Bruker, Germany) using deuterated water as solvent in a concentration of 10–15 mg/mL. The integral of the signal corresponding to the dPG protons 3.1–4.4 (m, 5H) with respect to the signals of the 3 protons of the vinyl group 5.98–6.12 (m, 1H), 6.18–6.32 (m, 1H), 6.45–6.53 (m, 1H) was used to obtain the degree of acrylation. The ratio between integrals of the signals evoked 2

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Ovalbumin and insulin were labelled with FITC for quantification purposes following manufacture protocols. In brief, 10 mg protein was dissolved in 1 mL of 0.1 M sodium bicarbonate buffer (pH 8.3–9.0). While stirring, 50–100 μL of FITC solution (10 mg/mL in DMSO) was added dropwise (~5 μL/min). The reaction was stirred for 1 h at room temperature under light protection. The separation of unreacted FITC was performed with a Vivaspin (MWCO 3 kDa) centrifugal devices centrifuging at 10.000 rpm for 10 min and washing with water. The proteins were lyophilized and stored at 6–8 °C. The labeling was confirmed by UV–Vis spectroscopy and the degree of labelling (DOL) was calculated according to reported protocol [37]. For Insulin DOL = 1.8–2.1 and for Ov-FITC DOL = 0.95–1.3. Protein encapsulation and in vitro release studies. To evaluate the release profiles of different molecular weight (Mw) proteins from the NGs the following proteins were assayed: Insulin human (6 kDa, 98%, Merck), Myoglobin from equine skeletal muscle (16 kDa, 95–100%, Merck), Ovalbumin (44 kDa, ≥98%, Merck) and Albuminfluorescein isothiocyanate conjugate (60 kDa, BSA-FITC, Merck). For protein encapsulation, 5 mg of lyophilized NGs were left to swell in 1 mL of a protein solution (PBS, pH 7.4) for 24 h at 6–8 °C. The NGs with the encapsulated protein were purified using a Vivaspin (MWCO 300 kDa) centrifuging 2 cycles at 6000 rpm of 10 min each. The encapsulated protein was quantified by UV–Vis spectroscopy by measuring absorbance at 492 nm for Insulin-FITC, Ovalbumin-FITC and BSA-FITC and at 409 nm for Myoglobin. The in vitro release experiments were performed in a Vivaspin device (MWCO 300 kDa). The NGs with the encapsulated protein were diluted with PBS to a final concentration of 1 mg/mL of NG and incubated at 25 °C and 37 °C. At determined time points, the samples were centrifuged (6000 rpm, 5 min), and the filtrate was analyzed by UV–vis spectroscopy and was replaced with the same volume of fresh buffer.

by the polyglycerol protons 3.1–4.4 (m, 5H) and by the polymeric backbone of PNIPAM (2.03 (1H) and 1.47 (2H)) and its isopropyl group 1.16 (6H) were used to determine dPG and PNIPAM content within the thermoresponsive NGs. Cloud point temperature (Tcp) measurements. Tcp values were determined by measuring the transmittance of a NG solution (2 mg/mL) at 500 nm during cycles of heating and cooling (20–60 °C, 0.5 °C/min) using a Cary 100 Bio UV–vis spectrophotometer equipped with a temperature controller sample-holder (LAMBDA 950 UV/vis/NIR, Perkin Elmer, USA). The Tcp is defined as the temperature of the inflection point of the normalized transmittance vs temperature of the heating curve. Particle size and ζ-potential. NG particle size, size distribution and ζ -potential were determined by dynamic light scattering (DLS) measurements using Malvern Zetasizer Nano-Zs 90 (Malvern Instrument, UK) equipped with a red He-Ne laser (λ = 633 nm, 4.0 mW) or a green DPSS laser (λ = 532 nm, 50.0 mW) at a scattering angle of 173°. Particle size and ζ -potential were measured in Milli–Q water and phosphate buffer, respectively. All samples were left to stabilize for 1–5 min at the certain temperature before being analyzed. For the determination of the volume phase transition temperature (VPTT) the size of the NGs was monitored using a heating rate of 1 °C/min. The VPTT is defined as the temperature of the inflection point of the normalized size vs temperature curve. Swelling ratio. The swelling ratio of each NG was calculated based on the volume ratio of the NG in swollen (25 °C) and collapsed state (T > VPTT). Every sample was done by triplicate. ANOVA and multiple comparison were performed under Fisher’s LSD method. 2.2.2. Synthetic protocols Synthesis of acrylated dPG (Ac-dPG). dPG of two molecular weights were used: 10 kDa dPG was obtained from Nanopartica GmbH (Germany) (PDI 1.3) and 5 kDa dPG was synthesized according to previously reported methodology (PDI 1.4) [36]. The acrylation was perfomed using pre-dried dPG (24 h at 80 °C under vacuum) and different amounts of OH were modified. The dry dPG (moles of OH to be modified, 1 Eq.) was dissolved in 20 mL of dry DMF and the solution was cooled down on ice. TEA (2 eq.) was added to the flask followed by dropwise addition of Ac-Cl (1.3 eq.) under argon atmosphere. The solution was stirred for 4 h and afterwards quenched by adding a small amount of water. Ac-dPG was purified by dialysis using a regenerated cellulose membrane (molecular weight cut-off (MWCO) 1 kDa) in water for 2 days. The product was obtained with a yield of 85–90% and stored at 6–8 °C. 1 H NMR (500 MHz, D2O), δ: 3.1–4.4 (m, 5H, polyglycerol scaffold protons), 5.98–6.12 (m, 1H, vinyl), 6.18–6.32 (m, 1H, vinyl), 6.45–6.53 (m, 1H, vinyl). Synthesis of dPG-NIPAM thermoresponsive nanogels. Thermoresponsive NGs were synthesized according to previously reported methodologies [4]. Different feed ratios of dPG (5 kDa or 10 kDa) and NIPAM were used to modify and control the final properties of the NGs. In brief, a total amount of 100 mg of the monomers (dPG and NIPAM) and SDS (1.8 mg) were dissolved in 4 mL of Milli-Q water. The reaction was purged with argon for 30 min and then transferred to an oil bath at 70 °C. After 15 min, a solution of KPS (3.3 mg, 1 mL) was added to initiate the polymerization. The reaction mixture was stirred for 4 h at 70 °C. The NGs were purified for 3 days via dialysis (regenerated cellulose, MWCO 50 kDa) in water. The product was lyophilized and rendered a white cotton-like solid with a yield of 80–90%. 1 H NMR of PNIPAM-dPG nanogels: (500 MHz, D2O), δ: 1.16 (s, 6H, isopropyl groups of NIPAM), 1.57 (2H, polymer backbone), 2.04 (1H, polymer backbone), 3.35–4.10 (6H, polyglycerol scaffold protons + 1H NIPAM). Protein labelling with Fluorescein 5-isothiocyanate (FITC).

3. Results and discussion 3.1. Effect of the acrylation degree and the crosslinker content 3.1.1. Synthesis and characterization of NGs The necessity to deliver a wide range of therapeutic molecules of different sizes highlights the need to generate a structural system that is able to be tuned according to the specific needs. In our previous articles, 10 kDa dPG was used as a crosslinker to generate NGs ranging in sizes between 100 and 200 nm [4], and were successfully used to deliver high molecular weight proteins through the skin, in a controlled manner [6,31]. However, there is still the lower part of the molecular weight range that we were not able to target as some of the proteins tend to diffuse out of the matrix. With the purpose of retaining small molecular weight proteins, we used 5 kDa dPG as a crosslinker to obtain dPG-NIPAM NGs with a denser structure. In this first part of the article, we focused on the control of the crosslinking density to evaluate their effect on the thermoresponsive properties and the network pore size. The use of dPG as crosslinker is based on its biocompatibility, highwater solubility and stability [38]. Moreover, its high-end-hydroxyl content enables to incorporate different functionalities to the system. Because of this potential multi-functionality we initially performed a screening on the amount of OH groups modified with vinyl groups, as can be seen in Table 1. dPG of 5 kDa with 2, 4, 7 and 13% of acrylic groups were obtained and used in a ratio of 30/70 wt% of dPG/NIPAM in the feed to synthesize the NGs. The degree of functionalization was calculated from a total amount of 67 mol of OH per mol of 5 kDa dPG by 1H NMR spectroscopy (Fig. S1). We found that dPG with an acrylation of 13% leads to aggregation of particles, while with lower acrylation percentage (2, 4 and 7%) stable nanogels were obtained (Table 1). The VPTT obtained by the curve size vs temperature remained invariable for all NGs with a value of 32.5 °C (Fig. 2A), but we did observe a trend on the swelling ratio by comparing their volume in 3

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Table 1 Monomer feed and characterization parameters of NGs synthesized using 5 kDa Ac–dPG (7%). The content of dPG and NIPAM was determined by 1H NMR. Size, PDI and ζ-potential are determined by DLS using intensity distribution curves. Nanogel

Degree of acrylation [%]

dPG:NIPAM feed

dPG:NIPAM content

Size [d.nm]

PDI

NG/5k/30 NG/5k/30 NG/5k/25 NG/5k/30 NG/5k/35 NG/5k/40 NG/5k/45 NG/5k/50 NG/5k/30

2 4 7

30:70 30:70 25:75 30:70 35:65 40:60 45:55 50:50 30:70

24:76 28:72 25:75 28:72 30:70 32:68 36:64 42:58 –

189 ± 14 210 ± 8 183 ± 13 166 ± 11 146 ± 5 132 ± 2 105 ± 7 71 ± 7 –

0.19 0.15 0.19 0.16 0.18 0.17 0.19 0.16 –

Size [nm]

A

13

2% Ac 4% Ac 7% Ac

150 100 50 25

30

35

40

45

50

Temperature [°C]

Swelling ratio

B 10 8 6

**

4 2

7

4

0 2

0.05 0.01 0.03 0.04 0.02 0.01 0.04 0.03

Yield [%]

−1.450 −0.960 −0.689 −1.236 −1.123 −0.936 −0.635 −1.369 –

85.4 87.2 87.5 88.1 84.2 85.1 75.1 72.1 –

decrease of the primary PNIPAM chain length. A similar behavior was previously reported for NGs using 10 kDa dPG as crosslinker [6,34,39]. Theune et al. also provided an insight comparing the system with lower molecular weight crosslinkers claiming that the high hydrophilicity of the dPG could help stabilizing the NG during the synthesis rendering in smaller NGs when compared to the ones synthesized with bis-acrylamide that has the opposite trend [6,40]. It was also noticed a decrease in the incorporated dPG vs the feed when the amount of crosslinker was increased (Table 1). While for lower dPG feeding ratios, feed and final content are the same, the increase of the dPG feed results in lower incorporations, e.g. when 50 wt % of dPG was added only 42% was incorporated to the actual NG. Additionally, the increase of the crosslinker amount was reflected on a decrease of the yield of the reaction. From these results, we can infer that when the amount of crosslinker is increased, larger amounts of oligoradicals could be formed, followed by the growing of the particles, but with a low NIPAM content some of the particles won’t be able to grow. These low molecular weight particles and un-reactive material could then be lost in the purification step upon dialysis rendering in a lower yield. These results are equivalent to the findings reported by Liu et al. for the synthesis of bis-acrylamide/PNIPAM NGs using high concentration of initiator that led to the formation of great amounts of low molecular weight radical precursors registering a drop in the yield after purification steps [41]. To understand the effect of the crosslinker content on the NGs thermoresponsiveness, the NGs Tcp was determined. Fig. 3B shows the temperature dependence of the normalized transmittance of the different NGs solutions. It can be clearly seen, that the Tcp did not vary with the crosslinking amount and remains around 33.5 °C. For the NG/ 5k/50, we observed that the transition is less pronounced and this same effect was also found when measuring the NGs size as a function of the temperature (Fig. 3C). The NGs synthesized with the lowest content of NIPAM (NG/5k/50) were less responsive to the temperature visible in only minor size change upon heating. This loss of the thermoresponsive behavior could be due to an increase on the rigidity of the network due to the combination of two factors: the increase of dPG amount will provide more crosslinking point that could generate a denser network and the decrease on the NIPAM content will create shorter polymer chains between the crosslinker points reducing the flexibility of the structure. In order to validate this statement, we analyzed the NGs swelling ratio as an indirect measurement of the flexibility of the matrix (Fig. 3D). A decrease on the swelling ratio was in fact observed when increasing the amount of dPG, this crosslinker dependency behavior can be supported by the Flory’s theory [42]. A polymer with a low crosslinking density can experience an abrupt phase transition due to a lower elasticity component. When the crosslinking density is increased, the competition between solvency and elasticity is more pronounced resulting in an increase of the rigidity of the system. Similar finding were reported for PNIPAM hollow microgels using N,N′-methylene bis(acrylamide) (BA) as crosslinker. The authors proved though atomic force microscopy that using high amounts of BA (10% and 17.5%)

250 200

± ± ± ± ± ± ± ±

ζ-potential [mV]

Acrylation of dPG [%] Fig. 2. Effect of the acrylation degree on (A) size dependency on temperature variation and on (B) swelling ratio of NGs with 2, 4 and 7% of acrylation.

swollen (below the VPTT) and shrunken state (over the VPTT). When the amount of acrylic groups are increased, more PNIPAM chains can be connected to the same dPG molecule in a way that the network becomes denser and less flexible, and these features are reflected in a reduction of the swelling ratio (Fig. 2B). A similar behavior was recently reported for the 10 kDa dPG, showing aggregation over 13% of acrylation and a decreasing in the swelling ratio with an increase of functionalities [6]. It is worth to mention that the swelling ratio of the 10 kDa NGs reported with 4 and 7% of acrylation are significantly higher than our findings suggesting that the use of smaller crosslinker could in fact have an impact on the network density, this behavior will be further discussed in the following section. In order to obtain a denser network to facilitate the encapsulation of smaller proteins we selected the dPG with 7% of acrylation for further study of the crosslinking density. For this, the feed amount of the monomers was systematically modified to modulate the final properties of NGs. Table 1 summarizes the reagent feed and the final proportion of dPG and PNIPAM in the NGs determined by the integral ratio of the 1H NMR signals corresponding to each monomer (Fig. S2). A linear dependency of the crosslinker content on the size of the nanogels was observed (Fig. 3A). A higher amount of crosslinker yielded in smaller NGs that could indicate a denser structure as a consequence of the 4

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B

200 150

Size [nm]

1.0

Normalized transmittance

A

100 50 0 20

25

30

35

40

0.8

NG/5k/25 NG/5k/30 NG/5k/35 NG/5k/40 NG/5k/45 NG/5k/50

0.6 0.4 0.2 0.0

45

25

NG/5k/25 NG/5k/30 NG/5k/35 NG/5k/40 NG/5k/45 NG/5k/50

100

40

45

50

8

Swelling ratio

Size [nm]

D

200

150

35

Temperature [ºC]

Crosslinker amount [wt%]

C

30

6 4 2

50

Temperature [°C]

50

0

50

45

45

40

40

35

35

30

30

25

25

Crosslinker feed amount [wt%]

Fig. 3. Effect of the crosslinker content. (A) Size of the NGs (B) Normalized transmittance curves (C) size dependency on temperature variation and (D) swelling ratios of the different thermo-responsive NGs.

matrix due to an increase in PNIPAM content might result in a greater deformation of the NG and this could be reflected into a higher incorporation of protein. Additionally, we observe a trend regarding protein size. In Fig. 4A it can be observed that Ins is incorporated in a greater amount into the NGs; in fact, for NG/5k/40 and NG/5k/30 it is still on its linear stage, while for Myo and Ov of the same NGs are already reaching the plateau for concentrations higher than 1.25 mg/mL of protein. These results infer on the capability of the protein to penetrate the NG, so smaller proteins can penetrate the network easier and accommodate better inside the NG structure compared to bigger proteins. Release profiles were taken as an indirect measurement of the network density and also to evaluate the potential as release system triggered by temperature. For this purpose, 5 mg of NG was dissolved in 1 mL of 0.5 mg/mL of protein, after purification, the solution was diluted to 1 mg/mL of NG using buffer PBS pH 7.4. Two different temperature conditions were evaluated. NGs were incubated at 25 °C to assess protein diffusion out of the swollen NG and at 37 °C, to evaluate the temperature-triggered release induced by the collapse of the nanogels. Fig. 5A shows the release profiles of the three proteins from NG/ 5k/50, NG/5k/40 and NG/5k/30. In all cases, a burst release of the protein was observed upon incubation of the NGs at temperatures above their VPTT (37 °C), in consequence of the collapse of the NG with the simultaneous release of water and the proteins. In contrast, at temperature below the VPTT, only minor amounts of the protein is released due to the diffusion through the pores. Comparing the release profiles for the three proteins, we found that the smaller the protein the faster is released to the medium (Fig. 5A). For example, after 1 h of incubation over the VPTT, 0.061 ± 0.008 mg of Ins was released, followed by Myo with 0.037 ± 0.003 mg released and finally, Ov only reaching 0.024 ± 0.001 mg for the case of NG/ 5k/30. It is worth to mention that this trend in which, for the same period of incubation, higher amount of Ins was released, followed by

prevented the microgel from total shrinking upon sample drying due to the high rigidity of the system. Whether the use of smaller percentages the hollow structure were not able to avoid a total collapse observing significant differences on the height of the microgels [43]. 3.1.2. Protein encapsulation and release The use of NGs systems for drug delivery applications requires a detailed knowledge of the network pore size in order to control the molecular exchange of a drug or protein. One effective approach to control the pore size is by tuning the crosslinking density. To evaluate how this parameter affects the network structure we have chosen globular proteins with different molecular weights for encapsulation and release experiments. To avoid differences in the electrostatic interaction with the NGs, the proteins should additionally have comparable isoelectric points (IP). We therefore selected Insulin (Ins) with a molecular weight of 6 kDa and an IP of 5.30, Myoglobin (Myo, 16 kDa, IP 6.8) and Ovalbumin (Ov, 44 kDa, IP 5.1). Additionally, as all NGs have a slightly negative to neutral ζ-potential value at pH 7.4 (Table 1), there will be no electrostatic interaction between the NGs and the proteins so we can expect that physical diffusion is the main contribution for the encapsulation process. To evaluate the impact of the crosslinking density on the matrix structure we selected from the previous characterized NGs three systems with different swelling ratio. We used NG/5k/50, NG/5k/40 and NG/5k/30 for loading and release experiments. Fig. 4A and 4B show the loading capacity and encapsulation efficiency of the selected NGs for all three proteins. We can see that while the loading capacity increases with higher protein/NG ratio, the encapsulation efficiency decreases rapidly. Overall, the loading capacity was considered high for all systems but NG/5K/30 was significantly higher for all encapsulated proteins, followed by NG/5k/40 and finally NG/5k/50 which exhibited the lowest values. These results might underline the importance of the network structure on the NG behavior, the higher flexibility of the 5

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Fig. 4. Protein encapsulation in three NG systems NG/5k/50, NG/5k/40 and NG/5k/30 with different protein concentration. (A) Loading capacity [%] and (B) encapsulation efficiency [%].

Here, we present the efficacy of three systems able to encapsulate low and high molecular weight proteins but differing on the release profiles. On one hand NG/5k/50 is able to release most of the cargo and on the other hand NG/5k/30 can retained most of the protein and this, could eventually find great potentiality as a reservoir system for long term release applications.

Myo and then Ov was observed for all NG systems incubated at 37 °C and 25 °C. Due the fact that the release of the proteins can be influenced by their size we can therefore conclude that the main mechanism involved in the release is the ability of the proteins to diffuse through the NGs pores. Additionally; a second trend was observed in correlation with the crosslinker content of the NGs. Paradoxically, higher dPG content led to a greater amount of protein released. NG/5k/50 exhibited the highest amount of released protein; this includes the temperature-triggered release due to collapse of the NGs at 37 °C and the release at 25 °C where the mechanism is solely based on diffusional processes. To our surprise, with each increment on the PNIPAM content, the diffusion rate of all proteins was further decreased at 25 °C as well as at 37 °C. This finding made us assume that the release mechanism could be more related to the length of the PNIPAM chains and its effect on the collapse of the system rather than a formation of a denser network. With very short PNIPAM chains between the dPG crosslinks, the temperature-induced conformational change will be less pronounced due to steric hindrance of inter-polymeric interaction by dPG. This would explain the scarcely change in the size for NG/5k/50 after incubation at temperatures above the VPTT shown in Fig. 3C. With the network structure barely changing above the VPTT, we assume that most of the pores will maintain ‘open’ with a similar pore size allowing more protein to diffuse out of the network. On the other hand, if the PNIPAM chains connecting the dPG are longer with fewer crosslinking points, as it is the case for the NG/5k/30, most of the protein was found retained within the NGs after the collapse (Fig. 5B). We can assume, that the proteins that are near the surface might be able to diffuse simultaneously with the expulsion of water from the NGs yielding the burst release profile at elevated temperatures. But the proteins that further penetrated into the NG might not be able to diffuse after the shrinkage of the network that induce a size-reduction and ‘closing’ of the pores. In this case, the pores sizes seem to be sufficiently big for the protein to penetrate during the encapsulation where the NG is in a completely swollen state, but become so small in the collapse state that most of the protein is retained. These findings highlight the relevance of understanding the network structure and how it influences the release mechanism that will have a great impact on the final application strategy.

3.2. Effect of the variation of the crosslinker size on protein release In order to understand how the molecular weight of the crosslinker affects the network structure of the NGs we decided to compare the NG/ 5k with a well-established model. In this matter, NG/10 kDa/30 (30/70 dPG 10 kDa/NIPAM) has been successfully used to encapsulate and release proteins with high molecular weight like BSA (66.5 kDa) [6,31] transglutaminase-1 (90 kDa) [31], and etanercept [33], and can serve as a study model to compare the network structure of both systems. For this, 10 kDa dPG was used as a crosslinker to obtain NGs. We used the same percentage of acrylation (7%) and the same weight monomer proportion (30/70 wt%) for the synthesis of NG/10 k/30 NGs (Fig. S3). Using the same weight percentage of dPG means that for the NG/10 k/ 30, half amount in moles was used of the crosslinker when compared to the NG/5k/30 NGs but as each molecule of 10 kDa dPG will have double the amount of vinyl groups, the moles of crosslinking points will be comparable between the two systems. A schematic representation of both NGs is illustrated in Fig. 6A. To our surprise, both NGs had comparable sizes (Table 2) with theoretically, the same crosslinking density. However, the swelling ratio of the NG/10 k/30 NGs is significantly higher than the NGs obtained with 5 kDa dPG (Table 2). The fact that both NGs had the same size and PNIPAM content but half the molecules of crosslinker in the case of 10 kDa dPG/NIPAM, might result in longer PNIPAM chains between the dPG molecules that could provide more flexibility to the network of the NG. This can explain why the NGs can shrink to a smaller size over the transition stage, as it is shown in Fig. 6B. Encapsulation and release experiments were performed using Ins, Myo, Ov and BSA. Fig. 7A shows the encapsulation efficiency of NG/ 10 k/30 and NG/5k/30 for all the proteins. The encapsulation efficiency in both NGs is comparable for each evaluated protein reaching in 6

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Fig. 6. NG/10 k/30 and NG/5k/30 (A) Schematic representation of the NGs and (B) size dependency on temperature variation.

In Fig. 7B and C it can be seen that Ins, the smallest protein, was released faster, followed by Myo, Ov and finally BSA. By comparing both NGs systems, we can observed that the incubation at 25 °C and 37 °C results in higher protein release when 10 kDa dPG was used as a crosslinker. By evaluating the diffusion of the protein at 25 °C (Fig. 7B) one might infer that the use of 10 kDa dPG would result in a less dense network allowing more proteins to diffuse out through the pores of the NGs. When the systems were placed over the transition temperature at 37 °C (Fig. 7C) to trigger the release we observed a similar trend as we did for the 25 °C, all proteins tend to be more retained in NG/5k/30. Giving the possibility of the connecting PNIPAM chains being shorter in the NG/5k/30 we can assume that during the collapse, the pores would rapidly be closed hindering the release of the proteins and retaining most of the cargo (Fig. 7D). In contrast, by evaluating the properties of NG/10 k/30 we can assume that has a more flexible network, giving by a higher swelling ratio, and a more open structure, observed by the higher diffusion of proteins at 25 °C. By these results we could deduce that the PNIPAM chains in NG/10 k/30 are longer and that during the collapse it would take more time for the network to reach the conformational state in which the proteins are no longer able to diffuse out. The versatility of the dPG/PNIPAM system relies on the careful selection of the starting materials and its final proportion in the reaction. This allow us to tune the structure of the network according to the desire application. The use of 10 kDa dPG as crosslinker and PNIPAM as connecting chain in a final proportion of 30/70 (dPG/NIPAM) is wellestablished system proven to be an effective release system trigger by temperature. Here, we demonstrated that the use of a smaller crosslinker (5 kDa dPG) and NIPAM in the same proportions would render in a more dense structure. The ability of this resulting NG to retained greater amounts of protein even in the collapse state could increase the

Fig. 5. (A) Release profiles of proteins and (B) amount of proteins retained in the NG after 24 h of incubation.

all cases 85–98%. Thus, we next evaluated the release of the proteins from both NG systems. We found the same trend for the release profiles correlated to the size of the proteins as discussed above (Section 3.1.2). 7

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Table 2 Monomer feed and characterization parameters of NGs synthetized using 5 kDa and 10 kDa dPG.

NG/5k/30 NG/10 k/30

dPG/NIPAM feed

dPG/NIPAM content

Size [nm]

PDI

Swelling ratio

30:70 30:70

28:72 27:73

166 ± 11 171 ± 6

0.16 ± 0.04 0.20 ± 0.01

5.1 ± 0.6 11.2 ± 0.1

and 10 kDa systems, had comparable sizes but different network flexibility giving by the higher swelling ratio of NG/10 k/30. We considered the possibility that having half molecules of dPG but the same amount of NIPAM could result in a NG longer PNIPAM chains between the crosslinkers. This difference in the network flexibility also allowed us to control the release profile of the proteins. In addition, this ability of the NGs to be deformed makes them with great properties to cross different biological barriers that includes skin [44], hair follicle and mucosal tissue [45]. We consider, in the future to have a special attention regarding the high retention of proteins inside de 5 kDa dPG NGs over the transition temperature as these NGs can be promising candidates to be used as reservoirs systems for long term release.

range of application to a long term release as a reservoir systems.

4. Conclusions In this article, we presented a rational design for the development of NGs with controllable size and release profiles. In order to obtain NGs with high density structure for the retention of small proteins we used dPG as crosslinker with a molecular weight of 5 kDa and PNIPAM as thermoresponsive connecting chains. A systematic screening of different feed ratio of the monomers was performed showing excellent tunability regarding NG size and swelling ratio. Proteins of different sizes were used as an indicative of the network density proving high encapsulation efficiency for all the systems. A correlation between the protein size and its release was observed, in which the smallest protein was able to diffuse through the pores in higher amounts. In addition, we proved that the release profiles can be easily tuned by the amount of crosslinker in the NG. Thus, the highest release correspond to NG/5k/ 50 NG that have the highest dPG content and a more rigid network with little volume change over the transition temperature. Increasing the NIPAM amount in the feed translated into a higher protein retention after the collapse of the NGs. In addition, to understand the effect of the molecular weight of the crosslinker on the network structure we used 10 kDa dPG as a well-established system for comparison. Both, 5 kDa

Data availability The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study. However, data will be made available on request. CRediT authorship contribution statement Lucila Navarro: Conceptualization, Methodology, Validation, Investigation, Writing - original draft, Visualization. Loryn E. Theune:

Fig. 7. (A) Encapsulation efficiency of proteins in NG/10 k/30 and NG/5k/30, (B) protein release at 25 °C, (C) protein release at 37 °C and (D) protein retained after incubation for 24 hs at 25 and 37 °C. 8

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Conceptualization, Methodology, Validation, Writing - review & editing. Marcelo Calderón: Conceptualization, Resources, Writing review & editing, Supervision, Project administration, Funding acquisition.

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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We gratefully Bundesministerium für NanoMatFutur award Foundation for Science, I00.

acknowledge financial support from Bildung und Forschung (BMBF) through the (13N12561), from IKERBASQUE- Basque and from MINECO project RTI2018-099227-B-

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