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Effective Drug Carrier Based on Polyethylenimine-functionalized Bacterial Cellulose with Controllable Release Properties Xiao Chen, Xuran Xu, Wenping Li, Bianjing Sun, Jun Yan, Chuntao Chen, Jian Liu, Jieshu Qian, and Dongping Sun ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00004 • Publication Date (Web): 22 Jun 2018 Downloaded from http://pubs.acs.org on June 23, 2018

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ACS Applied Bio Materials

Effective Drug Carrier Based on Polyethylenimine-functionalized Bacterial Cellulose with Controllable Release Properties Xiao Chen1†, Xuran Xu1†, Wenping Li1, Bianjing Sun1, Jun Yan3, Chuntao Chen1, Jian Liu3*, Jieshu Qian1,2*, Dongping Sun1*

1

Institute of Chemicobiology and Functional Materials, School of Chemical

Engineering, Nanjing University of Science and Technology, 200 Xiao Ling Wei, Nanjing 210094, China. 2

Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, School

of Environmental and Biological Engineering, Nanjing University of Science and Technology, 200 Xiao Ling Wei, Nanjing 210094, China. 3

Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University,

199 Ren Ai Road, Suzhou Industrial Park, Suzhou 215123, China †

These authors contribute equally to this work.

∗

Corresponding authors:

J. L.: [email protected], Tel.:+86-512-65884565; J. Q.: [email protected], Tel.: +86-25-84315173; D. S.: [email protected], Tel.: +86-25-84315079.

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ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract The development of low-cost biological materials with controlled drug release profile is of great importance but challenging in pharmaceutical industry. Recently, bacterial cellulose nanofibers have provoked intensive research interests in tissue engineering and the pharmaceutical science due to their stability, availability, sustainability and low toxicity. Here we describe the development of a PEI (polyethylenimine)-grafted bacterial cellulose (BC) as an efficient drug delivery system. The PEI-BC aerogels were characterized by SEM, FTIR, XPS, TGA and zeta potential measurements. The optimum sample exhibited enhanced mechanical strength, remarkable adsorption capacity towards aspirin, BSA and gentamicin, prolonged and pH-dependent drug release and low cytotoxicity. Our work has presented a rational structure design from biomass for controllable drug carrier.

Keywords Bacterial cellulose, three-dimensional network, polyethylenimine, aspirin, controlled release


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1. Introduction

Researches have put a lot of efforts to the design of effective drug delivery systems with high selectivity, biocompatibility and controlled drug release profile.1-3 Through releasing drug over a prolonged time period, an extended therapeutic effect may be realized by a sustained drug delivery system.4-5 Nonetheless, the challenges to obtain a sustainable drug effect still remain due to the low drug adsorption, limited biological activity and specific release environments such as the stomach or intestine.6-7 Efforts have been made on the design of sophisticated drug formulations using various nanomaterials, such as nanoparticles, nanocapsules8, micelles9, and aerogels10. Among them, aerogels are getting increased attention as drug carriers due to their open porous structure, high added-value lightweight and high surface area, which guarantee a good adsorption and retention of water within their three-dimensional network without dissolution.11 The most used classes of aerogel material for such purpose are silica, carbon and polysaccharide.12-14 The bio-based polysaccharide polymers, such as starch15, cellulose16, chitosan17 and alginate18 have been widely used as key formulation ingredients for drug delivery systems on account of their high stability, low level of toxicity and enhanced drug loading capacity.19 Recently, cellulose nanofibers (CNF) have provoked an increasing interest in the applications of tissue engineering and pharmaceutical science.20 The Ivanov group employed CNF-titania nanocomposites as carrier for three types of medicines, diclofenac sodium, penicillamine-D and phosphomycin. Their results demonstrated a great potential of the CNF-based nanocomposites for transdermal drug delivery or wound-dressing.21 In another example, the Svagan group fabricated CNF-based foams with different thicknesses and sizes for the loading of riboflavin. The materials showed a prolonged release in simulated gastric fluid (SGF) and its potential as a sustained drug delivery system.22 Moreover, the Zhang group synthesized

PEI-grafted

bamboo

nanofibrils

which

exhibited

pH-

and

temperature-responsiveness to sodium salicylate with a drug loading capability as high as 287.39 mg g-1, exploiting the application value of cellulose in drug delivery.23 Bacterial cellulose synthesized from Acetobacter xylinum has drawn an increasing ACS Paragon Plus Environment


attention and interest in the field of biomedical device due to its unique structure and properties.24-25 The high purity, excellent mechanical strength together with large surface area could guarantee strong drug-CNF interaction, which improves their potentials as adsorption materials, drug carriers as well as biological scaffolds.26 Nonetheless, the fact that BC is used as an excipient in pharmaceutical formulations is not widely acknowledged in public journals.27 For instance, the Yang group investigated the controlled release behavior of BC membranes for loading berberine hydrochloride and berberine sulphate, which showed the lowest release rate in SGF and the highest in simulated intestinal fluid (SIF).28 The Amin group examined the function of BC-g-poly-(acrylic acid) hydrogels as proteins carrier and demonstrated only 10 % cumulative release in SGF.29 They also reviewed the studies focused on the use of BC films to modulate drug release in transdermal systems, however, the low drug loading capability of pristine BC in the form of film may limit its further commercialization. Other studies focused on the property enhancements of BC and biomedical polymer composites, but their further application in drug absorption and release was not mentioned.30 Consequently, it is vital to develop BC materials with enhanced loading capacity and sustained drug delivery. Herein, we report a design of a controlled drug release system using BC nanofibers as scaffold. The BC nanofibers were first oxidized by TEMPO to form oxidized CNF, followed by a crosslinking reaction with branched PEI. A subsequent freeze-drying treatment of the PEI-BC generated aerogels, which were further used as an effective vehicle to encapsulate protein and model drugs under different conditions. We also investigated the influence of PEI contents on the loading capacity. Our results have demonstrated a potential use of BC nanofibers in biomedical application.


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2. Experimental 2.1 Materials BC nanofibers were cultivated using an Acetobacter xylinum NUST4.2 through a dynamic fermentation process at 30 °C.24 After the fermentation, the sample was treated with 0.1 % sodium hydroxide solution and hydrogen peroxide at 80 °C for 6 h in order to remove the bacteria. Then the nanofibers were washed with distilled water several times until a neutral pH value. All other materials used in this study are described in Supporting Information (SI). 2.2 Synthesis of TEMPO-BC 5.0 g BC was suspended in a 0.1 M sodium acetate buffer solution (pH = 4.5) in a breaker. The oxidation was performed at 25 °C with the addition of 75 mM TEMPO and 8 U m L−1 laccase. After stirring for 16 h, the mixture was under an oxygen pressure of 0.6 MPa for 8 h. Afterwards, the oxidized BC was extensively washed with distilled water by repeated centrifugation and re-dispersion in water. The amount of carboxylic groups was determined by conductimetric titration. 2.3 Synthesis of PEI-BC-x A series of PEI-BC-x aerogels was prepared from a series of 100 mL 2 % w/v TEMPO-BC aqueous solutions containing different amounts of PEI. The value of x represents the weight ratios of PEI to BC to be 0.5:1, 1:1, 2:1 (denoted as PEI-BC-0.5, PEI-BC-1, PEI-BC-2). 0.25 g EDC and 0.25 g NHS were added into each solution for the crosslinking reaction under stirring at 25 °C for 24 h. The obtained PEI-BC gels were washed with distilled water to remove residual reagents. Finally, they were transferred into a 24-well plate and freeze-dried for 48 h. 2.4 Drug Loading Studies. In order to investigate the effects of PEI contents as well as reaction time, a series of batch adsorption experiments was carried out through static adsorption. All the uptake experiments were conducted by adding 15 mg adsorbents (cylinder with height of 2.0 cm and diameter of 2.0 cm) into 20 mL drug solutions at designated concentrations ranging from 100 to 3000 mg L-1 for 24 h. For the PEI-BC-2 sample, BSA and gentamicin loading experiments were also performed in PBS buffer under pH = 7.4 to ACS Paragon Plus Environment


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investigate the multifunctional adsorption capacity. Meanwhile, the adsorption kinetic was performed by mixing 15.0 mg PEI-BC-2 sample into 50 mL 500 mg L-1 drug solutions. At time t, 0.5 mL solution was removed from the mother solution to examine the drug contents by a UV-vis spectrophotometry at wavelengths of 280 nm31 and 232 nm32 for BSA and gentamicin, respectively. The adsorption capacity (AC) of aerogels could be calculated via equation 1: AC =

(1)

where Co and Ct (mg L-1) represent the initial concentration and concentration at time t, m (g) and V (L) are the weight of the adsorbent and the volume of the solution. 2.5 Drug Release Studies. The release behavior was evaluated on different pH environments. 30 mg Aspirin-loaded PEI-BC-2 aerogel (cylinder with height of 2.0 cm and diameter of 4.0 cm) was suspended in 50 mL SGF (0.2 % NaCl, pH = 2.5) and 50 ml SIF (0.05 M, NaH2PO4, pH = 7.5) at 37 °C. The experiments were carried out on a rotary shaker at 100 rpm for 24 h. At time t, 2 mL solution was taken out to determine the aspirin contents by UV-vis spectrophotometer at a wavelength of 276 nm. Meanwhile, 2 mL fresh SGF or SIF solution was added to maintain a constant volume. The cumulative percentage (CP) of drug release was calculated via equation 2: CP =

∗ 100%

(2)

where Co and Ct (mg L-1) represent the initial concentration and the concentration at time t. 2.6 Characterization FT-IR spectra were collected on a Thermo Scientific Nicolet iS5 spectrometer with scanning wavelengths from 500 cm-1 to 4000 cm-1. The ζ-potential measurements were performed by a Malvern Nano-ZEN3500 Zetasizer. The XRD patterns were obtained by using a Bruker AXS D8 advanced diffractometer with Cu Kα radiation (λ = 1.5418 Å) at 40 kV and 35 mA. XPS spectra were collected using an RBD-upgraded PHI-5000C ESCA system (Perkin Elmer) with Mg K radiation (h = 1253.6 eV). The elemental analysis was carried out using a Costech ECS 4010


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analyzer. Scanning electron microscopy (SEM) images were collected using a Hitachi S-4800 operating at 25 kV. All samples were coated with a thin layer of evaporated gold before testing. Thermogravimetrical analysis (TGA) was carried out on a METTLER TOLEDO TGA/SDTA851 thermogravimetric analyzer at a heating rate of 10 °C min-1(50-700°C, nitrogen atmosphere). 2.7 Cytotoxicity Tests of the Materials. TEMPO-BC and PEI-BC-2 were rinsed with alcohol and deionized water in turn and exposed to ultraviolet radiation for 2 h before cell seeding. The drug loading concentration was 5 mM for the two samples. After sterilization, the samples were pre-treated by immersing in Dilbecco’s Modified Eagle Medium (DMEM) in 96 well plates for 24 h. 4T1 (mouse breast tumor cell), HepG2 (liver hepatocellular carcinoma), 293T (kidney epithelial cell), and HUVEC (human umbilical vein endothelial cells) were seeded at a cell density of 1.5 × 104 cells per well with 10 % sample concentrations in DMEM. After 48 h of incubation, CellTiter-Glo assay was performed to determine the cell viability value by Veritas microplate luminometer (Promega).33



3. Results and discussion

Scheme 1. Experimental design of the PEI-BC crosslinking composites.

The key novelty of this paper is the design of multicomponent aerogels using the BC three-dimensional network as scaffold to combine a functional PEI polymer via chemical crosslinking, as illustrated in Scheme 1. The interpenetrated network of multicomponent aerogel after crosslinking normally presents better physico-chemical properties than its constituents.34 In our design, the BC nanofibers were oxidized to turn the surface C6 primary hydroxyl groups to carboxylic acid groups,35 allowing the subsequent amidation reaction with the amino groups of PEI. The abundant amino groups on PEI make it popular for the adsorption of various substances especially for environmental application36-37 as well as for gene delivery.38 The oxidation system of TEMPO/laccase/O2 rather than the traditional TEMPO/NaClO/NaBr was utilized since the former could provide a milder and more eco-friendly environment, while the later generates a byproduct NaBrO to depolymerize and deteriorate the CNF.39 It is important to produce carboxylic acid groups on the CNF surface because the electrostatic repulsions of carboxylic acid groups could inhibit fiber aggregation, which is a common issue for CNF.40 In SI, we describe the conductimetric titration experiments and calculations to evaluate the contents of the carboxyl groups. Based on the titration curves for sample BC, TEMPO-BC and PEI-BC-2 in Figure S1 in SI (discussion also presented in SI), a content (0.86 mmol g-1) of carboxylic acid groups was determined for TEMPO-BC.


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Characterizations of Materials.

Figure 1. SEM images of (a) BC, (b) TEMPO-BC, (c) PEI-BC-0.5, (d) PEI-BC-1, (e) PEI-BC-2 aerogels and (f) optical image of TEMPO-BC and PEI-BC-x samples before and after weight loading.

The morphologies of BC, TEMPO-BC and PEI-BC-x samples were first examined by SEM and shown in Figure 1a-e. The TEMPO-BC and PEI-BC-x samples retain the three-dimensional interconnected porous structure as BC. The pristine BC is observed to possess the highest density of the interconnected structure due to the aggregation of CNF. The TEMPO-BC sample possesses the largest pores with sizes from 20 to 300 µm. In PEI-BC-x samples, one could see glue-like structure to cross-link the CNF and the formation of irregular two-dimensional sheets. As the amount of PEI (value of x) increases, the density of the interconnected structure seems to increase and the pore walls become thicker. In Figure 1f, we present two sets of optical images of TEMPO-BC and PEI-BC-x samples before and after weight loading. Under the same weight condition, both TEMPO-BC and PEI-BC-0.5 undergoes an obvious deformation, while PEI-BC-2 almost preserves the original form. These results suggest that the PEI cross-linking changes the morphologies of BC and endows the interpenetrated networks of CNF with higher mechanical strength. The chemical structures of BC, TEMPO-BC and PEI-BC-2 were then investigated by



FT-IR (Figure 2a). The spectra of the PEI-BC-x series are shown in Figure S2 in SI. Compared to the pristine BC sample, the new peak at 1636 cm-1 for TEMPO-BC is ascribed to the C=O vibration of the carboxylic acid groups. As for PEI-BC-2, new peaks at 1645, 1560, 1459 cm-1 appear. The absorption peak at 1645 cm-1 could be related to the C=O stretching of the amide group. 41 The peaks at 1560 and 1459 cm-1 correspond to the amino group of PEI.42 Moreover, the band found between 3200 cm-1 and 3600 cm-1 is associated with the vibration of amine N–H group, which becomes stronger with the increasing of PEI (value of x) content as shown in Figure S2.

Figure 2. a) FT-IR spectra, b) XPS spectra and c) TGA curves of BC, TEMPO-BC and PEI-BC-2 aerogels, and d) Zeta potential of TEMPO-BC and PEI-BC-x samples.

The chemical states of these samples were further characterized by XPS. The spectra of BC, TEMPO-BC and PEI-BC-2 are shown in Figure 2b, the spectra of the PEI-BC-x series are shown in Figure S3 in SI. From these figures, one could clearly see the appearance of N 1s peak at 398-402 eV in PEI-BC-x samples, which does not


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exist in BC and TEMPO-BC. As the amount of PEI (value of x) increases, the intensity of N 1s peak increases significantly. The PEI-BC-2 sample has the highest nitrogen content, which is estimated to be ca. 17 %. The results from the elemental analysis experiments of each sample are summarized in Table S1 in SI. The difference in the nitrogen content between the PEI-BC-x and TEMPO-BC further supported the FT-IR and XPS results of the presence of PEI in BC. The thermal decomposition of the composite aerogel was characterized by TGA analysis. The weight loss curves of BC, TEMPO-BC and PEI-BC-2 are shown in Figure 2c. The pristine BC displays a typical decomposition temperature range from 280 °C to 389 °C, which is much higher than CNF from plant cellulose.35,43 The residues of BC at 700 °C was higher than that of TEMPO-BC (24 % compared to 18 %), suggesting a lower thermal stability of TEMPO-BC due to the existence of carboxylic groups. As for PEI-BC-2, two new degradation stages are observed besides the typical cellulose pyrolysis. The first stage from 142 °C to 246 °C is attributed to the decomposition of the amino groups in PEI macromolecule chains, while the second from 374 °C to 425 °C is due to the decomposition of the polymer backbone, which is in good agreement with other reported PEI films.44 Notably, PEI-BC-2 was thoroughly decomposed at a higher temperature in comparison with those of TEMPO-BC. The chemical functionalization of BC would inevitably alter its charge properties. Thus, the zeta potential measurements were conducted on TEMPO-BC and PEI-BC-x samples. The results are shown in Figure 2d, where TEMPO-BC displays an isoelectric point of 2.3 on account of the introduction of carboxylate groups on BC surface. In contrast, PEI-BC-x samples display much larger values of 8.3-9.4. The value of isoelectric point of the PEI-BC-x series increases as the increase of the PEI content. These values are mainly influenced by the abundant amino groups of PEI in the samples.45

Adsorption kinetics of PEI-BC-x Aerogels toward Aspirin. In this section, we describe the adsorption properties of the PEI-BC-x aerogels. An ACS Paragon Plus Environment


important drug aspirin was used as a prototype to evaluate the adsorption kinetics of the three PEI-BC-x aerogels. The adsorption kinetics data are shown in Figure 3 and fitted by three different models, i.e. a pseudo-first order model (Eq. 3, Figure 3a), a pseudo-second order model (Eq. 4, Figure 3b) and an intra-particle model (Eq. 5, Figure 3c): ln = ln

=

!

= "# $.& ! '

(3) (4) (5)

where qe and qt (mg g-1) are the adsorption capacities of aspirin at equilibrium and at time t (h), k1 (min−1) is the rate constant of the pseudo-first order model, k2 (g mg−1h−1) is the rate constant of the pseudo-second order model, kid (mg g−1 h−0.5) is the intra-particle diffusion rate constant and C (mg g−1) is the thickness of the boundary layer. The correlation parameters calculated from the fitting are summarized in Table S2 in SI.

Figure 3. Adsorption kinetics of PEI-BC-0.5, PEI-BC-1 and PEI-BC-2 towards aspirin at 25 °C, data fitted by (a) a pseudo-first order model; (b) a pseudo-second order model and (c) an intra-particle diffusion model.

Figure 3a shows that for all samples, the adsorption takes place rapidly in the beginning and reaches more than 80 % of the maximum uptake within 5 h. The adsorption equilibrium could be attained within 10 h. From the values of fitting parameters in Table S2, where one sees that the pseudo-second order model (Figure 3b) is more applicable for the adsorption data than the pseudo-first order model with higher values of correlation coefficients R2. The adsorption capacities at equilibrium


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are determined to be 212 mg g-1 (PEI-BC-0.5), 246 mg g-1 (PEI-BC-1) and 345 mg g-1 (PEI-BC-2), respectively. It is not surprising to see the remarkable improvement of adsorption capacity of PEI-BC versus BC (5 times improvement, see Figure S4 in SI for details) due to the presence of PEI. With respect to the data fitting by the intra-particle diffusion model in Figure 3c, one could see three distinct regions, corresponding to three steps of the diffusion of aspirin to PEI-BC-x aerogels, i.e. (I) external film or boundary layer diffusion; (II) macropore or region diffusion; and (III) micropore diffusion.46 The fact that the second linear region of macropore diffusion is clearly observed is consistent with the macroporous structure of PEI-BC-x aerogels

Multi-drug Adsorption of PEI-BC-2 Aerogel. Since multi-drug delivery for proteins and/or antibacterial purpose is generally required in tissue engineering,47 we then investigated the adsorption properties of our PEI-BC-2 sample towards several guest molecules, i.e. aspirin, BSA (Proteinic drug) and gentamicin (SAIDS, steroidal anti-in-flammatory drug). PEI-BC-2 was chosen as the representative of the PEI-BC-x series since it possesses the highest content of PEI and has shown the highest adsorption capacity towards aspirin in the previous section. The adsorption isotherms as well as kinetics are shown in Figure 4. All isotherms are fitted by Langmuir (Eq. 6) and Freundlich (Eq. 7) models: =

( )* +)*

= ,- '. /0

(6) 7

where Ce (mg L-1) is the equilibrium concentration of drugs in solution, qe (mg g-1) is the adsorption capacity at equilibrium, qm (mg g-1) is the maximum adsorption capacity, KL (L mg-1) and KF (mg g-1 (L mg-1)1/n) are constants for Langmuir and Freundlich isotherms, and n is a Freundlich constant relating to adsorption intensity of the adsorbents. The corresponding parameters calculated from the equations are summarized in Table S3 in SI. The adsorption isotherms fitted better to the Langmuir model than the Freundlich model with much higher R2 values, indicating a monolayer adsorption and the homogeneous surface of PEI-BC samples.48 We calculated the



maximum adsorption capacities of PEI-BC-2 towards aspirin, BSA and gentamicin to be 309 mg g-1, 74.0 mg g-1 and 20.9 mg g-1, respectively. The adsorption kinetics data in Figure 4 show that for all three drugs, PEI-BC-2 could achieve ca. 80 % of the maximum uptake within 5 h. The adsorption equilibrium could be reached in 15 h for all samples. These results have demonstrated an excellent adsorption ability of PEI-BC-2 towards several model drugs. In Table S4 in SI, we show the comparison of our sample with other similar drug carrier as reported previously. Our PEI-BC-2 aerogel has shown its great potential as drug carrier in biomedical applications due to the excellent adsorption capability.

Figure 4. Adsorption isotherm and kinetics of PEI-BC-2 at 25 °C towards (a,b) aspirin, (c,d) BSA and (e,f) gentamicin. Adsorption isotherms are fitted by both Langmuir and Freundlich models. Drug Release Studies.


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In this section, we describe the results showing the release profile of aspirin by the PEI-BC-2 aerogel in comparison with TEMPO-BC. The plots of cumulative release of aspirin versus time under two conditions, i.e. SGF (pH = 2.5) and SIF (pH = 7.5), are presented in Figure 5a. The aspirin release rate is calculated using a pseudo-second-order equation (eq 8): 2

=

2

!

2

(8)

where Qe (%) is the cumulative release at equilibrium, Qt (%) is the cumulative release at time t, and k2 (h−1) is the rate constant of pseudo-second-order equation. The TEMPO-BC aerogel shows a quick release of aspirin in the very beginning, reaching the equilibrium within 30 min. However, PEI-BC-2 shows a much slower release rate, reaching the equilibrium within ca. 10 h under both conditions. These results suggest that the PEI grafting greatly improves the sustainable drug release behavior of BC aerogel probably due to the electrostatic adsorption between the amino groups and aspirin. Figure 5b shows that the aspirin release kinetics could be well fitted by a pseudo-second order model (R2 > 0.99) and the experimental data are fairly closed to the estimated values. The fitting parameters are summarized in Table S5 in SI. It is also interesting to see that the drug release behavior of PEI-BC-2 is greatly affected by the solution pH. In SIF (pH = 7.5), the sample exhibits a cumulative aspirin release of 80.6 %, which is much higher than the 52.3 % in SGF (pH = 2.5) of the same sample. We emphasize that in real clinical practice, this pH-sensitive behavior could lessen the release of aspirin in stomach and reduce the negative effects on stomach. Meanwhile, the absorption efficiency of aspirin in intestine could be enhanced. These results have demonstrated the prolonged drug release performance as well as the remarkable pH responsiveness of our PEI-BC-2 aerogel. Additionally, we evaluated the release profile of PEI-BC-2 in two more pH conditions (4 and 9). The results are summarized in Figure S5 and Table S5 in SI.



Figure 5. (a) Aspirin cumulative release curves versus time and (b) data fitted by a pseudo-second-order model of BC and PEI-BC-2 aerogels at pH = 7.5 and pH = 2.5. Cytotoxicity Tests. In the end, we show the results of cytotoxicity test of our materials on different cell lines in Figure 6. A set of blank control experiments was carried out on the standard tissue culture plates (TCPs) for comparison and normalization. One can see that the normal cell lines 293T and HUVEC maintain nearly 90 % of cell viability on the substrates of TEMPO-BC and PEI-BC-2, indicating the low cytotoxicity of our samples. After drug loading, the cell viability has slightly decreased, but still remains above 80%. However, due to the anti-cancer therapeutic effect of aspirin, the cell viability decreases significantly after drug loading for the two cancer cells lines 4T1 and HepG2. Meanwhile, the drug loaded PEI-BC-2 showed a stronger inhibition of cancer cells than that of TEMPO-BC, which is probably due to the improved drug loading capacity and sustainable drug release behavior of PEI-BC-2 as we have shown in the previous sections.


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Figure 6. Cell viabilities on TCPs, TEMPO-BC, PEI-BC-2, TEMPO-BC with aspirin and PEI-BC-2 with aspirin of (a) 293T cells (b) HUVEC cells (c) 4T1 cells (d) HepG2 cells at culture times of 24 h and 48 h.

4. Conclusion In summary, we report a design of aerogels as sustained drug carriers from biomass bacterial cellulose. A functional PEI polymer with abundant amino groups was chemically cross-linked to the modified BC three-dimensional interconnected network. The obtained PEI-BC aerogels possess higher mechanical strength and more macropores than the pristine BC. Remarkably, our best sample exhibited the maximum loading capacities of 309 mg g-1, 74.0 mg g-1, and 20.9 mg g-1 towards aspirin, BSA and gentamicin, respectively. Meanwhile, our materials showed a prolonged and pH-dependent release of aspirin. With the virtues of high loading



capability, controllable release profile and good biocompatibility, our PEI-BC aerogel has shown its potential as during carrier for further pharmaceutical applications. We also hope that our work could stimulate more work towards the development of biomaterials based drug loading systems with simple structure design.

Supporting Information Materials, determination of the contents of carboxyl groups of the materials; FT-IR and XPS of PEI-BC-0.5, PEI-BC-1 and PEI-BC-2 aerogels; Elemental analysis results of BC, TEMPO-BC, and PEI-BCs; Adsorption isotherms of PEI-BC-2 and TEMPO-BC towards Aspirin; Aspirin cumulative release of BC and PEI-BC-2 aerogels at pH = 4 and pH = 9; Fitting parameters of adsorption kinetics data towards aspirin, the adsorption isotherms towards aspirin, BSA and gentamicin to Langmuir and Freundlich models and release kinetics of TEMPO-BC and PEI-BC-2 towards aspirin with different pH values; Comparison of the adsorption performances of various adsorbents towards aspirin, BSA and gentamicin.

Acknowledgements The authors acknowledge financial support from the National Natural Science Foundation of China (51572124, 21504045, 51573079, and 51702162), Fundamental Research Funds for the Central Universities (30916012201, 30915012202, and 30916014102) Nanjing University of Science and Technology, Qing-Lan Project from Jiangsu Education Department, and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, China). Postgraduate Research and Practice Innovation Program of Jiangsu Province.


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