Lotus Leaf-Inspired Bone Cement Particles with Ultrahigh Drug

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Lotus leaf-inspired bone cement particles with ultrahigh drug encapsulation capacity Botao Song, and Gaoli Hu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00115 • Publication Date (Web): 22 Jun 2018 Downloaded from http://pubs.acs.org on June 23, 2018

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ACS Applied Bio Materials

Lotus leaf-inspired bone cement particles with ultrahigh drug encapsulation capacity Botao Song*, Gaoli Hu Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi'an 710069, Shaanxi, People’s Republic of China. Correspondence to: Botao Song (E-mail: [email protected])

Abstract: Nowadays interest is growing toward drug-loaded inorganic particles with high drug encapsulation capacity to treat bone disease. However, the conventional adsorption method and in situ co-precipitation method always encountered the problem of low drug encapsulation efficiency. The phenomenon of water droplet standing in a near-spherical shape with very small contact area on the lotus leaf inspires us to demonstrate a simple and green strategy to handle this issue. Briefly, the slurry containing bone cement powders, drug and water is firstly dripped onto the superhydrophobic surface to form a microreactor, then the drug will be encapsulated in the confined microreactor due to the self-setting reaction of the bone cement powders with water. Calcium sulfate hemihydrate (CSH), a kind of biocompatible and historical bone cement, is employed to prepare calcium sulfate dihydrate (CSD) bone cement particles. Two kinds of drugs acetaminophen and ibuprofen with different water solubility are further individually embedded into the CSD matrix to investigate the drug encapsulation and release behaviors. The results indicate that the CSD particles with tunable diameters can be easily prepared by changing the volume of the dispensing droplets. Most importantly, the drug encapsulation efficiencies can reach up to 91% for both the hydrophilic and the hydrophobic drug, which are the highest as compared with the other reported methods. It is also indicated that the drug release profiles can be finely controlled by the size of the CSD particles and the drug loading amount. Our strategy provides a novel pathway in the rational design of drug-loaded particles with ultrahigh drug encapsulation efficiency, which shows great advantages in the bone repairing applications.

Keywords: bone cement, calcium sulfate, superhydrophobic, drug encapsulation efficiency, control release

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Introduction Inorganic particulate drug delivery system is regarded as an effective route to treat bone diseases, which can not only control the drug release in the target site, but also can be used as bone filling materials to repair bone defects1-3. In addition, the voids between the inorganic particles are favor for the new bone formation and new vascular ingrowths4. Considering the merits of the inorganic particulate drug delivery system, a variety of approaches such as adsorption method and in situ co-precipitation method have been proposed to fabricate such kind of drug delivery system. The main strategy of the adsorption method is to select biocompatible materials with porous structure, including mesoprous silica, mesoporous bioactive glass, porous hydroxyaptite, to adsorb drugs from the drug solution into the nanopores or micropores of the porous materials5-7. Unfortunately, the drug will be inevitablely lost into the liquid environment, thus, the drug encapsulation efficiency is not optimal. The procedure of washing the drug adsorbed on the out surface of the porous carrier will further decrease the drug encapsulation efficiency. The in situ co-precipitation method is also employed to fabricate drug-loaded particles, in which the formation of the drug carriers and the drug encapsulation are accomplished simultaneously in the same reaction system8-10. Since the drug is inclined to diffuse into the reaction solution, the drug encapsulation efficiency is also not satisfied. Therefore, the common reason for the low drug encapsulation efficiency of these methods is that the whole preparation process is carried out in the liquid phase, thus abundant drug will be lost into the solvent but not be encapsulated efficiently within the drug carriers. The low drug encapsulation efficiency may cause severe drawbacks, such as decreasing the bioavailability of the drug, leading to low drug concentration in the target site, increasing the patients’ burden especially for the expensive drugs (the anticancer drugs, the biomacromolecule drugs, and so on). Therefore, it is highly desirable to develop a novel method to fabricate inorganic particulate drug delivery system with ultrahigh drug encapsulation efficiency. Recently, “lotus leaf effect” inspires numerous researchers to fabricate superhydrophobic surface for biomedical applications, such as anti-biofouling, disease diagnosis, prevention of blood adhesion on the blood-contacting implantable medical devices, and drug sustained release11-18. For example, Zhang and coworkers reported a novel superhydrophobic polyorganosilanes modified halloysite nanotube for controlled release of diclofenac sodium, due to the air cushion between the


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superhydrophobic nanocontainer and the buffer solution, the superhydrophobic gated nanocontainer exhibited a prolonged drug release behavior16. Owning to the distinct wetting behaviors between the superhydrophobic and superhydrophilic surface, Levkin et al ingeniously fabricated a miniaturized platform based on the superhydrophobic-superhydrophilic micropatterns for high throughput cell screening18. The key point is that this kind of special wettable surface shows strong repellency to water due to the entrapped area layer within the superhydrophobic surface, which leads to a very small contact area between the liquid and the superhydrophobic surface. Shall we drip water droplet containing drug onto this surface? It is speculated that by virtue of the strong anti-adhesive property of the special wettable surface almost all the drug will be embedded within the water droplet, thus, high drug encapsulation capacity can be easily achieved. Unfortunately, as it is known that it is impossible to directly use the water/drug droplet to treat disease, because it is untouchable and unmanufacturable. The self setting reaction of inorganic bone cement powders with water provides a new pathway to settle this issue19-21; hence, we further consider to utilize bone cement powders to solidify the water/drug droplet, because this kind of materials can harden into the solid form once contacting with water, therefore, the drug can be stably and efficiently embedded. Besides, the low adhesion of the superhydrophobic surface allows the drug-loaded particles to be easily detached and ready to use. The fabrication procedures of drug-loaded bone cement particles are illustrated in Figure 1. We firstly electrospray the fluropolymer solution onto the metal substrate to fabricate a superhydrophobic surface, which we have reported previously22. Calcium sulfate hemihydrate (CaSO4·1/2H2O, CSH) is a kind of historical bone repairing material due to its excellent biocompatibility, biodegradability, and osteoconductivity23-26. Previous studies also demonstrated that the calcium sulfate was an ideal drug carrier material27. Besides, the CSH can be quickly converted into calcium sulfate dihydrate (CSD) when mixing with water due to its excellent self-setting property28. All these features motivate the fabrication of CSD bone cement particles on the superhydrophobic substrate. Prof. João F Mano’s group utilized the superhydrophobic substrates to prepare polymer beads for loading drugs, and the polymer beads exhibited high efficiency in drug encapsulation29,30. The main topic of this study was to construct drug-loaded particles for bone repairing. Generally, for particle based bone fillers in bone tissue regeneration, three major issues should need to be resolved: (1) high drug encapsulation efficiency and controllable release, (2) osteoconductivity, (3)



and good mechanical property. A number of problems would be encountered regarding the use of polymer beads in bone regeneration application. For example, the mechanical strength of the polymer hydrogel beads was compromised after filling into the bone defects. Moreover, the polymer beads were always lack of osteoconductivity. Besides, for preparing polymer beads UV light must be used for crosslinking, and the unreacted residual monomers might decrease the biocompatibility of the polymer beads. All these limited their application in bone regeneration. By contrast, in this study we selected bone cement material CSH to fabricate inorganic particles due to its excellent biocompatibility, osteoconductivity, and good mechanical property. In addition, water was only required for the solidification of the CSD particles on the superhydrophobic substrate, and the fabrication process did not need UV light and no residual toxic chemicals were involved. All these features motivated the fabrication of inorganic CSD inorganic particles for bone repairing, which had lots of merits than polymer beads. Therefore, in this study the controllable fabrication of CSD particles with different sizes is carried out, and two different kinds of drugs with different water solubility are chosen as model drugs to investigate the drug encapsulation and release profiles of the CSD bone cement particles.

Figure 1. Schematic illustration of the process for the fabrication of drug-loaded bone cement particles on a superhydrophobic substrate.


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Materials and methods

Materials CSD was purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd. Ibuprofen (IBU) was purchased from Tokyo Kasei Kogyo Co. N,N-dimethylformamide (DMF) was purchased from Tianjin Tianli Chemical Reagents Co., Ltd. Acetaminophen (AMP) was supplied by Aladdin Reagent Company. All the reagents were used as received without further purification.

Fabrication of the superhydrophobic surface In a typical synthesis procedure, 3 mL of 15 mM 2, 2'-azobisisoheptonitrile ethanol solution was slowly poured into a three-necked flask at 61°C under N2 environment. Sequently, a mixture of 6 mL 2, 2'-azobisisoheptonitrile ethanol solution, 50 mL of dodecafluoroheptyl methacrylate solution with the concentration of 0.2 g/mL and 50 mL of 2-hydroxyethyl methacrylate solution with the concentration of 0.04 g/mL was slowly added in the above solution for 3 hour. After vigorous stirring for 24h, the final fluoropolymer was obtained. The NMR spectrum of the fluoropolymer was measured, which was coincided with our previous result22. The superhydrophobic surface was prepared according to our previously published paper22. Typically, 0.1315 g fluoropolymer was dissolved into 3.0 mL of DMF and continuously stirred for 1 h to obtain a homogenously transparent solution. Then, the cleaned aluminium plate was immersed into DMF for 2 min and further fixed tightly on a conductive collector by the conductive adhesive for the electrospray process. The voltage about 10 kV was applied between the collector and the metal needle. The feeding rate of the solution was 1.5 mL/h. The electrospray process was carried out at room temperature and the relative humidity was about 55%. The fluopolymer coated aluminium plate was then dried at ambient environment for 24 h.

Fabrication of CSD particles with different sizes The CSD particles were fabricated with the conversion of CSH into CSD on the superhydrophobic surface. Firstly, the CSH powders were synthesized according to a previously published paper31. Typically, CSD powders were dried at 150 °C for 12 h, followed by transferring into the oven with the temperature of 60 °C for 6 h. Sequentially, the CSH powders were added into distilled water with the liquid to solid ratios of 1.5 mL/g to form a slurry. In order to minimize the lost of CSH



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powders and drugs, the slurry was stirred in a fluorinated ethylene propylene beaker. The slurry with precise volume was then dripped onto the superhydrophobic surface by using a pipette. After then, the droplets would gradually transform into CSD particles due to the self setting reaction. In order to fabricate CSD particles with different sizes, the slurry droplets with different volume (4, 6, 8, 10 µL) were carefully transferred by a pipette onto the superhydrophobic surface, and the corresponding sample names were denoted as S1, S2, S3, and S4, respectively. The method for fabrication of drug-loaded CSD particles was the same as that of the pristine CSD particles. Two kinds of model drugs hydrophilic drug AMP and hydrophobic drug IBU were selected to investigate the drug loading and releasing of the CSD particles. Typically, for fabrication of AMP-loaded CSD particles, 6.75 mg AMP was dissolved into 0.45 mL of distill water with vigorously stirring to obtain a homogeneous solution. Then, 0.3 g CSH powder was quickly mixed with AMP aqueous solution to form a slurry, followed by dripping onto the superhydrophobic coating with the pipette. The AMP-loaded CSD particles were gradually formed. Due to the hydrophobicity of the model drug IBU, the fabrication process of IBU-loaded CSD particles was different from that of AMP-loaded CSD particles. The IBU was firstly fully mixed with CSH powders, then the mixture was vigorously stirred in the distill water to form a slurry. IBU-loaded CSD particles were prepared by dripping the slurry on the superhydrophobic surface. In order to adjust the drug release, drug-loaded CSD particles with different sizes and different drug loading amount were all fabricated. The detailed composition of the drug-loaded CSD particles was listed in Table 1.

Table 1. The composition of the slurry and the volume of the droplet dripped on the superhydrophobic substrate. Sample

Droplet CSH (g)

Water (mL)

AMP (mg)

IBU (mg)

name

volume (µL)

S1

0.3

0.45

0

0

4

S2

0.3

0.45

0

0

6

S3

0.3

0.45

0

0

8

S4

0.3

0.45

0

0

10


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S5

0.3

0.45

1.69

0

4

S6

0.3

0.45

1.69

0

6

S7

0.3

0.45

1.69

0

8

S8

0.3

0.45

1.69

0

10

S9

0.3

0.45

3.38

0

10

S10

0.3

0.45

6.75

0

10

S11

0.3

0.45

0

1.69

4

S12

0.3

0.45

0

1.69

6

S13

0.3

0.45

0

1.69

8

S14

0.3

0.45

0

1.69

10

S15

0.3

0.45

0

3.38

10

S16

0.3

0.45

0

6.75

10

In addition, the drug-loaded CSD particles were also fabricated on different substrates to investigate effect of wetting behaviors of the substrates on the drug encapsulation and controlled release. Four kinds of substrates (glass; stainless steel; polystyrene; superhydrophobic substrate) were firstly employed, and they were cleaned by the acetone and distilled water. Then, the slurry and the drug mixture were carefully dripped on the substrates, after the self-setting process, the particles were carefully taken down. The composition of the slurry was referred to sample S8.

Characterization The morphology of the samples was analyzed by using a scanning electron microscopy (SEM, S-4800, HITACHI, Japan). The sizes of the CSD particles were measured by a vernier caliper. Typically, 30 CSD particles were randomly selected, and both the width and the height of the particles were accurately measured, and the corresponding height/width ratio was also calculated. The water contact angle (WCA) of the four kinds of substrates was investigated by using a Data Physics OCA 20 Tensiometer at 25 °C. A 5 µL water droplet was carefully dripped onto the substrate, and the average WCA value was obtained by measuring ten different positions on the substrate. Similar procedures were also employed to investigate the formation process of the CSD particles on the superhydrophobic substrate. The contact angle hysteresis (CAH) was determined



by using the Wilhelmy plate method with a DCAT 21 tensiometer (DataPhysics Instruments GmbH). The detailed procedures were carried out according to Gong’s publication32. The CAH was the difference between the advancing and receding angle.

The compressive strength of the CSD particles The compressive strength of the four kinds CSD particles was measured by a die-plunger method according to the previous publications33,34. Typically, the particles were manually poured into a hollow cylindrical container, after carefully shaking to make the particles close packing, then a plunger was slightly put into the container. The test was carried out using a UTM 2103 electronic universal testing machine.

The change of particle size in PBS We further measured the change of particle size during immersion in PBS at 37 °C. Before incubation, the morphology of the CSD particles was firstly tested by the optical microscopy, and the initial size of the particle was carefully measured based on the scale bar within the optical image. Then, the particles were incubated into the PBS, at the predefined time points, the samples were taken out, dried, and both the width and height of the particles were further carefully measured. The change of both the width and height of the particles was calculated according to the following equation: Change of the size (%) = (Do-Dt)/Do×100% Where Do and Dt represented the original size (width and height) of the sample and the size of the sample after soaking in PBS for different time, respectively. Experiments were run in triplicate per sample and the results were expressed as mean ± standard deviation.

The drug encapsulation efficiency and in vitro drug release To evaluate the drug encapsulation efficiency, ten drug-loaded CSD particles were dissolved into 10 mL of hydrochloric acid solution (1 mol/L) for 24 h. The concentration of AMP and IBU was analyzed by using a UV-Vis spectrophotometer at the wavelength of 242 and 264 nm based on the standard curves, respectively. The drug encapsulation efficiency was calculated using the following equation:


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The drug encapsulation efficiency=m2 / m1×100%. Where m1 denoted the weight of drug initially added in the slurry, m2 was the weight of the drug embedded within the CSD particles which was calculated according to the standard curve of the drug concentration versus absorbance. Each experiment was run in triplicate per sample and the results were expressed as mean ± standard deviation. To study the drug release kinetics, the drug-loaded CSD particles were placed into 20 mL of PBS at pH 7.4 and vibrated in the shaker at 37 °C. At the certain time intervals, 5.0 mL of the soaking solution was withdrawn to measure the released drug concentration and the same volume of fresh PBS solution was added to keep the volume constant. Both the concentration of AMP and IBU that remained in the buffer solution were determined using a UV-Vis spectrophotometer, respectively. The cumulative release percentage was calculated based on the standard curve, and the results were expressed as mean ± standard deviation. All the release experiments were repeated for three times.

Results and discussion



Figure 2. (A) Digital image of colourful water droplets resting on the superhydrophobic substrate (Insert showed the SEM image and the WCA of the superhydrophobic substrate); The transition of CSH droplets into CSD particles on the superhydrophobic substrate (B:before self setting; C: after self setting); (D) The formation process of the CSD particles minitored by a contact angle meter; (E) The CSD particles shaking in the PBS with the rotation speed of 90 rpm for 12 h.

In the present study, superhydrophobic substrate is vital for the fabrication of drug-loaded bone cement particles, therefore, we firstly used a very simple and versatile electrospray method to prepare such special wettable surface. It was found that all the colourful water droplets labelled


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with different dyes exhibited near-spherical morphology and the WCA of the substrate was about 153.46° (Figure 2A), indicating the superhydrophobic state of this substrate, which was in coincided with our previous results22. The SEM result demonstrated that the coating was consisted of lots of interconnected particles, which could greatly increase the roughness of the substrate. Both the rough surface structure and low surface energy composition of numerous inherent -C-F groups endowed the coating with superhydrophobic property. The CAH was further measured to be about 5.54°. Such a small angle indicated a low adhesion force of the bone cement slurry on the superhydrophobic surface, thus during the drying process the three-phase contact line would gradually decrease, and only a small contact area was existed between the bone cement particle and the superhydrophobic substrate, which was very vital for the high drug encapsulation efficiency. Besides, the very low CAH also indicated the easy rolling of the particles from the superhydrophobic substrate, thus the collection of the particles became more convenient. Overall, the low CAH was of great significance in preparation of drug-loaded particles on superhydrophobic substrate. The effect of liquid to solid ratio (L/S) on the formation of CSD particle was firstly investigated, and it was found that the optimal L/S should be 1.5 mL/g. When the L/S was lower than 1.5 mL/g, the slurry was highly viscous, which could hardly be dripped by the pipette. By contrast, when the L/S was higher than 1.5 mL/g, the droplet on the superhydrophobic surface consisted of abundant water, and the resulted particle was unstable and easily disassembled when incubating into PBS. Thus, the optimal L/S was 1.5 mL/g. As depicted in Figure 2B, the wet CSH slurry with the optimal L/S was dipsensed on the superhydrophobic surface, and it was observed that all the droplets were shown in the form of near-spherical morphology; sequently, due to the self setting process, the CSH droplets were gradually transformed into the dry solid CSD particles (Figure 2C). A contact angle meter was further employed to investigate the formation mechanism of CSD particles on the superhydrophobic surface. From the side view shown in Figure 2D, it was also observed that the slurry droplet was in a quasi-spherical morphology; more importantly, the contact area between the the CSH slurry and the substrate was very small; as the time increased, the three-phase contact line became shorter and the size of the CSD particle was gradually decreased; after 20 min the CSD particle was completely hardened, which could be proved by shaking into the PBS solution. After 12 h continous shaking no small CSD microparticles could be



observed within the solution (Figure 2E). The formation mechanism might be that the quasi-spherical droplet on the superhydrophobic surface acted like a microreactor. Two physicochemical changes were happened within the confined microreactor, one was the water evporation, and the other was the reaction of CSH with water (self setting behavior)35,36. Both the two reactions comsumed water, thus as time goes by the three-phase contact line became shorter and the size of the particle was gradully decreased.

Figure 3. Digital images and SEM images of the four kinds of CSD particles with dispensing different volume of slurry (A, B: S1; C, D: S2; E, F: S3; G, H: S4); (I) The height, (J) width, and (K) the height to width ratio of the four kinds of CSD particles.

Based on the above results, the CSD particles with controllable sizes were further synthesized by dispensing with different volume of slurry (4; 6; 8; 10 µL), and the morphology were characterized by both the digital camera and the scanning electron microscopy. As seen from the digital images (Figure 3A; C; E; G), it was clearly observed that lots of CSD particles with different sizes could be successfully fabricated by this approach due to the easy detach of the particles from the superhydrophobic substrate. The scanning electron microscopy was further employed to investigate the microstructure of the CSD particles. Surprisingly, the surface feature


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of the four kinds of CSD particles was apparent different. As depicted in Figure 3B, the surface of the sample S1 was consisted of lots of microparticles with different sizes. In contrast, the morphology of the sample S3 became relatively regular, and large crystal plate and the assembly could be observed (Figure 3F). As the volume of the slurry increased to 10 µL, the morphology of sample S4 became highly structured, and many nanoplate and nanorod assemblies were found (Figure 3H). Such morphology was the typical morphology of the CSD bulk material, where the CSD crystal could be fully grown due to the sufficient water. As mentioned above, the formation of CSD particles on the superhydrophobic surface was related to two reactions, one was the self-setting reaction of CSH with water, and the other was the water evaporation within the droplet. For the sample S1, after the droplet dripped on the superhydrophobic surface, the formation of the CSD phase was quickly occurred due to the fast self setting process of the CSH powders; simultaneously, the evaporation reaction was also happened, due to the very small volume of the droplet about 4 µL, the water in the droplet was easily evaporated, therefore, there was no sufficient water for the growth of the CSD crystal into a bigger size, which resulted in a small size of the CSD crystal. As the volume of the droplet gradually increased to 10 µL, abundant water was provided for the CSD crystal growth, which resulted in a full growth of the CSD crystals; thus, the CSD crystal became larger and more regularly. In addition, we also found that the required time for fabrication of the four kinds of CSD particles were obviously different, which was gradually increased from sample S1 to S4. The phenomenon further proved that as the volume of the droplet increased, the growth of the CSD crystal tended to be more sufficient and more organized. The sizes of the four kinds of CSD particles were carefully measured by a vernier caliper, and the corresponding height to width ratios were also calculated, shown in Figure 3(I, J, K). It could be clearly observed that both the height and width of the particles were gradually increased with the increasing of the droplet volumes. Moreover, it was also found that for all the samples the height was a little bit smaller than the width, which was caused by the gravity of the droplet on the superhydrophobic surface. In order to investigate the similarity of the morphology of the particles to a sphere, the height/width ratios of the CSD particles further were caculated. As it is known, when the height to width ratio was 1, and the resulted particle could be identified as a sphere. As depicated in Figure 3K, the height to width ratios of the four kinds of CSD particles were 0.87, 0.85, 0.73, and 0.77, respectively, which demonstarted that the prepared CSD particles were



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quasi-spheres. The above results indicated that both the macro and micro morphology of the CSD particles could be finely controlled by the volume of the droplet dripped on the superhydrophobic substrate.

Figure 4. (A) The compressive strength of the four kinds of CSD particles; the change of (B) height and (C) width of the CSD particles after immersion in PBS for 7 days.

We further measured the compressive resistance of the CSD particles by a die-plunger method, and the results indicated that no significant difference of the compressive strength was found for all the four kinds of CSD particles (Figure 4A). The compressive strength of our particles was higher than the reported calcium phosphate cement particles at the same strain34. The reason was that, as mentioned above the CSD particles were formed within the confined liquid droplets, thus the nanoplates and nanorods within the particles stacked densely. Besides, the resultant particles were highly uniform, which could be closely packed. Both the two reasons resulted in a relatively high compressive resistance of the CSD particles. Figure 4 (B, C) showed the change of particle size after soaking in PBS, and it could be seen that both the width and height of the particles were gradually decreased after soaking for 7 days, and only a slight difference was found among the four kinds of samples. Previous studies had proved that the CSD was a kind of degradable inorganic material. After incubation into PBS, the CSD particles were gradually degraded, thus the size of the particles became smaller.

Table 2. The WCA and CAH of the four kinds of substrates, and the corresponding drug encapsulation efficiency based on the four kinds of the substrates. Kinds of substrates

WCA (°)

CAH (°)


Encapsulation efficiency (%)

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Glass

23.64 ± 3.00

22.34 ± 2.24

47.19 ± 3.62

Stainless steel

75.97 ± 4.05

55.20 ± 1.16

57.60 ± 6.84

Polystyrene

112.51 ± 3.77

32.36 ± 0.73

66.03 ± 6.01

153.46 ± 2.64

5.54 ± 1.11

95.18 ± 2.45

Superhydrophobic substrate

It is known that bone defect is usually coexisted with lots of bone diseases, such as bone tumor, osteoporosis, bone tuberculosis, and so on. Therefore, the prepared particles should not only be used to fill into the bone voids to repair bone defect, but also should possess the capacity for controlled drug delivery to treat bone diseases. As mentioned above, high drug encapsulation efficiency was very important for the bone fillers. In order to clarify the significance of the superhydrophobic surface in drug encapsulation, four kinds of substrates with distinct wetting behaviors (glass; stainless steel; polystyrene; superhydrophobic substrate) were employed to prepare drug-loaded CSD particles, and both the drug encapsulation efficiency and release behaviors were systemically investigated. The wetting behaviors of the four kinds of substrates were firstly measured, and the WCAs were 23.64° for glass, 75.97° for stainless steel, 112.51° for polystyrene, and 153.46°for superhydrophobic substrate, and the CAHs were 22.34°, 55.20°, 32.36°, and 5.54°, respectively (Table 2). The drug encapsulation was further studied, and from the digital images it could be found that lots of residual bone cement powders were tightly stacked on the substrates (glass; stainless steel; polystyrene). In comparison, almost no residual could be found on the superhydrophobic substrate by the naked eye, which indicated the high drug encapsulation efficiency with the use of superhydrophobic substrate (Figure 5). The corresponding encapsulation efficiencies were further calculated, and the results indicated that the drug encapsulation efficiency was much higher by using the superhydrophobic substrate than that of other substrates (Table 2).



Figure 5. (A-D) The appearance of the four kinds of substrates after removing the drug-loaded CSD particles (A blue paper was placed under the transparent glass and polystyrene substrates for easy observing), and (E) the drug release profiles of the four kinds of drug-loaded particles.

The conventional substrates usually exhibited relatively small WCAs and large CAHs. The small WCA resulted in the easy spread of the slurry droplet on the substrate, that is, the contact area between the substrate and slurry droplet was very large. Besides, large CAH led to the tight pinning of slurry droplet on the surface. Both this two reasons led to large amount of CSD powders remaining on the substrate, which hence resulted in relatively low drug encapsulation efficiency. In this study, the superhydrophobic substrate we employed exhibited large WCA and low CAH, which efficiently decreased the contact area between the droplet and the superhydrophobic substrate, thus a very high drug encapsulation efficiency was achieved. The drug release profiles of the drug-loaded CSD particles fabricated on the different substrates were further studied, and an obvious two stages of drug release was found, that is, a burst release at the early stage followed by a sustained release in the late stage, shown in Figure 5E. After incubation in PBS for 5 days, about 88.3%, 85.0%, 82.2%, and 78.9% of the drug was released from the CSD particles fabricated on the different substrates (glass; stainless steel; polystyrene; superhydrophobic substrate). It could be concluded that the drug release profiles were influenced by the wetting behaviors of the substrates. During the fabrication of particles on the hydrophilic glass substrate, the slurry droplet was quickly spread out on the glass due to the highly hydrophilic of this substrate, thus the obtained drug-loaded CSD particles were in a form of sheets. Owning to the very thin thickness of the particles, the drug could be easily released into the buffer


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solution, which resulted in a fast release. As the substrate was more hydrophobic, the thickness of the drug-loaded CSD particles became large, and the release of drug became more difficult, which resulted in a relatively slow drug release. When choosing the superhydrophobic substrate as the platform a near spherical morphology could be obtained, the diffusion path of drug was longer, thus the release of drug could be retarded.

Figure 6. (A) The drug encapsulation efficiency and (C) release profiles of AMP-loaded CSD particles with different sizes (sample S5-S8) and different drug loading contents (sample S8-S10); (B) The drug encapsulation efficiency and (D) release profiles of IBU-loaded CSD particles with different sizes (sample S11-S14) and different drug loading contents (sample S14-S16).

Due to the merit of using superhydrophobic substrate in drug encapsulation, two model drugs with different solubility (AMP and IBU) were chosen to fabricate drug-loaded particles on the special wettable surface. Firstly, AMP-loaded CSD particles with different sizes (sample S5-S8) and different AMD loading ratios (sample S8-S10) were prepared and the drug encapsulation efficiency was systemically investigated, shown in Figure 6A. Interestingly, all the drug encapsulation efficiencies were higher than 92%; moreover, both the size of the CSD particles and



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the drug loading amount had no obvious influence on the drug encapsulation efficiency. Further, we tested the capacity of the CSD particles to load a hydrophobic drug IBU, and the results also indicated over 91% of IBU could also be embedded within the CSD particles (Figure 6B). That is, the lotus leaf-inspired method could efficiently embed both the hydrophilic drug and the hydrophobic drug. Further, the release of AMP and IBU from the drug-loaded CSD particles was investigated, respectively. As seen from Figure 6C, effect of particle size on the release profile of AMP was firstly evaluated, and it was found that the drug exhibited two stage of release. At the initial 24 h, the drug was released quickly and about 61.3%, 66.8%, 71.1%, and 72.7% of the drug were released, respectively; after then, the drug release tended to be stable, after 39 days of incubation, about 67.7%, 74.3%, 78.7%, and 81.2% of the drug were released, respectively. Further, the effect of drug loading content on the drug release was studied, and similar drug release kinetics were observed. After releasing of 39 days, a total about 81.2%, 84.6%, and 89.3% of the drug was released, respectively. From these data, it could be found that the release of AMP could be modulated by the size of CSD particles and the drug loading content. Similarly, we also observed that the release of hydrophobic drug IBU could be controlled by the size of CSD particles and the drug loading content, and the burst release of IBU was significant decreased compared to that of the AMP. The SEM results shown before indicated that all the obtained CSD particles had open pore structure, which made the drug AMP easily diffuse out from the pores into the release medium. Another reason was that the model drug AMP was highly water-soluble, which could be easily dissolved and diffused into the buffer solution. Thus, a quick release behavior was observed at the early stage of AMP release; by contrast, because the hydrophobic drug IBU was hard to dissolve into water, thus the release of IBU was significantly slower than that of the AMP.

Table 3. Comparison of drug encapsulation efficiency by the CSD bone cement particles and other drug delivery systems. No.

1

Drug carrier

mesoporous bioactive

Drug

Encapsulation

Reference

efficiency

number

63.6%

37

doxorubicin


Page 19 of 26 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


glass nanospheres 2

porous hydroxyapatite

gentamicin sulfate

30.9%-41.8%

38

ibuprofen

24.16%-27.58%

39

alendronate

65.9%-72.0%

40

dimethyloxaloylglycine

14.57% -21.79%

41

microspheres 3

porous hydroxyapatite microspheres

4

calcium phosphate microspheres

5

mesoporous silica nanospheres

42

calcium 6

phosphate-phosphorylated

doxorubicin

42.3%

imatinib

77.59%

acetaminophen

＞92%

ibuprofen

＞91%

adenosine microspheres 7

mesoporous bioactive

43

glass nanospheres 8

CSD particles

This study

In this study, we ingeniously proposed a lotus leaf-inspired approach which could efficiently embed different kinds of drugs, and the encapsulation efficiency could high up to 91% and the above, which was much higher than the reported drug delivery systems (Table 3). The conventional drug loading methods (adsorption method and in situ co-precipitation method) were always carried out in the liquid phase, and the drug would be inevitablely diffused and lost into the liquid environment, therefore the drug encapsulation efficiency was relatively low. In addition, the further washing process which was to remove the untightly adsorbed drug and the unreacted reagent further decreased the drug encapsulation efficiency. By contrast, in our study the entire drug embedding process was happened on the solid surface, and the possible location for the drug was in the particles or in the contact area. Due to the embedded air within the special wettable surface the contact area between the slurry and the surface was very small, thus almost all the drug would be encapsulated within the CSD particles. In addition, the prepared drug-loaded particles could be used directly without further washing, which decreased the possibility of drug loss. Both



the two reasons resulted in the high drug encapsulation efficiency of the drug carrier. Overall, compared with other conventional methods, this lotus leaf-inspired approach had the five following outstanding features. (1) High drug encapsulation efficiency. Due to the anti-adhesive property of the superhydrophobic substrate and the solid interface reaction, only a slight drug loss was happened during the drug encapsulation process. (2) Both hydrophilic and hydrophobic drugs could be efficiently encapsulated. (3) Not only calcium sulfate hemihydrate, other inorganic bone cement materials, such as calcium phosphate bone cement, calcium silicate cement, magnesium phosphate bone cement, could all be manufactured into the quasi-spherical shape to load drugs44-46. (4) The shape of the water droplet on the superhydrophobic surface guided the macroscopic strcture of the CSD particle in a near-spherial shape which could be used to fill into the irregular bone defects; moreover, the voids between the particles were favor for the ingrowth of the neotissues and new blood vessels4. (5) This method could fabricate bi-functional bone repairing materials with both the drug delivery capacity and bone filling property. We believe the development of such a bio-inspired approach will open up a wide range of research opportunities on bone regeneration.

Conclusions In summary, this study demonstrated a simple superhydrophobic surface-induced self setting process, that enabled the rapid production of drug-loaded bone cement particles with ultrahigh drug loading efficiency and controllable drug delivery property. By utilizing this method, drug-loaded CSD particles with different sizes and different drug loading amounts were successfully fabricated. Moreover, it was also found that both the hydrophilic drug and the hydrophobic drug could be well encapsulated within the CSD particles and the drug encapsulation efficiency could reach up to 91% and the above, which was much higher than the reported drug delivery systems. This novel approach would open a new avenue for the fabrication of bone regeneration materials.

Acknowledgements This research was supported by grants from Natural Science Foundation of China (No.: 31600767) and Top-rated Discipline construction scheme of Shaanxi higher education.


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Lotus Leaf-Inspired Bone Cement Particles with Ultrahigh Drug

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