The adsorption of tetracycline and vancomycin onto nanodiamond with controlled release

Journal of Colloid and Interface Science 468 (2016) 253–261

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The adsorption of tetracycline and vancomycin onto nanodiamond with controlled release James Giammarco a, Vadym N. Mochalin a,b, James Haeckel c, Yury Gogotsi a,⇑ a

Department of Materials Science and Engineering, A.J. Drexel Nanomaterials Institute, Drexel University, Philadelphia, PA 19104, United States Department of Chemistry, Missouri University of Science & Technology, Rolla, MO 65409, United States c Department of Chemical Engineering, A.J. Drexel Nanomaterials Institute, Drexel University, Philadelphia, PA 19104, United States b

a r t i c l e

i n f o

Article history: Received 27 October 2015 Revised 26 January 2016 Accepted 27 January 2016 Available online 28 January 2016 Keywords: Nanodiamond Adsorption Release Drug delivery Nanoparticle Tetracycline Vancomycin Antibiotics

a b s t r a c t The unique properties and tailorable surface of detonation nanodiamonds have given rise to an abundance of potential biomedical applications. Very little is known about the details of adsorption/desorption equilibria of drugs on/from nanodiamonds with different purity, surface chemistry, and agglomeration state. The studies presented here delve into the details of adsorption and desorption of tetracycline (TET) and vancomycin (VAN) on nanodiamond, which are critically important for the rational design of the nanodiamond drug delivery systems. The nanodiamonds studied in these experiments were as-received (ND), purified and carboxyl terminated (ND-COOH), and aminated (ND-NH2). The monolayer capacities of the drugs loaded onto the nanodiamonds are reported herein using Langmuir and Freundlich isotherm models. The results from the desorption studies demonstrate that, by changing the pH environment of drug loaded nanodiamond using buffers of pH 4.09, 7.45, 8.02, and a phosphate buffered saline (PBS) solution, the drug release can effectively be triggered. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction Nanocarrier drug delivery systems offer a wide range of controllable properties that can be used to facilitate therapeutic procedures [1]. Careful control is required to maintain the delicate balance between loading as much drug as possible (to increase efficiency) and loading too much drug so that it starts to leak from the nanocarrier before being delivered to a desired location. Undesired potential leaks of drugs from the nanocarrier can be minimized or prevented only when a delivery system is sufficiently understood. Potential leaks are of concern because many drugs used to treat illnesses or cancer have adverse effects on other parts of the body, kill normal cells, and over time can poison the body [2,3]. Furthermore, antibiotic drugs are becoming increasingly ineffective. This often leads to higher doses or eventually requiring a more potent (and often more toxic) drug. Vancomycin (VAN) is known to have Abbreviations: TET, tetracycline; VAN, vancomycin; ND, as-received nanodiamond; ND-COOH, carboxyl terminated nanodiamond; ND-NH2, amine terminated nanodiamond; PBS, phosphate buffered saline; MRSA, Methicillin-resistant Staphylococcus aureus; KCl, potassium chloride; BCA, bicinchoninic acid; FTIR, Fourier transformed infrared; WR, working reagent; ATR, attenuated total reflectance; HCl, hydrochloric acid; NLLS, non-linear least squares. ⇑ Corresponding author. E-mail address: [email protected] (Y. Gogotsi). http://dx.doi.org/10.1016/j.jcis.2016.01.062 0021-9797/Ó 2016 Elsevier Inc. All rights reserved.

high ototoxicity [3] to the body and is used as a last resort drug to kill resistant Gram positive infections such as MRSA [4,5]. Tetracycline (TET), a broad spectrum antibiotic [6] may not be acutely toxic compared to vancomycin but if stored improperly byproducts of its degradation can be toxic to humans [7]. TET has equally concerning environmental implications for aquatic environments [8]. For these reasons combined, over the recent years, there is an increasing interest in developing non-toxic and low-cost nanoparticle carriers for targeted delivery of toxic and poorly soluble drugs, including antibiotics, as opposed to their systemic administration [9,10]. Commercially produced detonation nanodiamond (NDs) particles have good biocompatibility, no reported toxicity, and a tailorable surface chemistry [11]. By attaching drugs to the surface of NDs and their controlled release, it is possible to renew the potency of anticancer chemotherapeutics [12]. Similarly, the development of NDs for targeted delivery of antibiotics and other drugs may also restore the drug efficiency and reduce systemic toxicity. Interest in ND for biomedical applications including biomedical imaging, tissue engineering, and drug delivery systems has been growing steadily. Currently, ND is being investigated for the delivery and sustained release of anticancer chemotherapeutics [12,13], nucleic acids [14,15], and insulin [16]. It has also been demonstrated for covalent binding of proteins, sometimes enhancing

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their activity [17]. However, our understanding of basic mechanisms and underlying principles for development of a better ND drug delivery system is still in rudimentary state [18]. Here we report results on the adsorption and pH dependent release of tetracycline and vancomycin, onto/from ND with different surface chemistries (Fig. 1c and d). Focus was on the monolayer capacity of adsorbed drug on the surface, and the binding strength of the drug. Antibiotics tetracycline and vancomycin were chosen because their efficiency to bacteria can potentially be restored through nanoparticle delivery systems. Adsorption/desorption is the preferred mechanism for any drug delivery system due to its relative simplicity, and no or minimal changes to a drug structure during adsorption. In this study, we aim to understand the basic mechanisms and effects of ND surface chemistry on adsorption and release of drugs, which will help to select ND with proper functionalization for each type of drugs. 2. Materials and methods 2.1. Materials and solutions Vancomycin hydrochloride 98% purity was purchased from Cayman Chemicals. Tetracycline hydrochloride 99% purity was purchased from Fisher Scientific. The drugs were used without further purification. Solutions of vancomycin and tetracycline were prepared in deionized water for calibration curves. Nanodiamond of UD90 grade was donated by SP3 and characterized elsewhere [19]. ND-COOH was synthesized by air oxidation of UD90 at 425 °C for 5 h and then purified from trace metals by

acid treatment for 24 h [19]. ND-COOH was aminated by linking ethylenediamine through amide bond, yielding ND terminated with NH2 groups (ND-NH2). The description of this wet chemistry amination procedure can be found elsewhere [20]. A bicinchoninic acid (BCA) protein assay kit was purchased from Fisher Scientific for analysis of vancomycin release only, as described below. Buffer solutions (pH 4.09, 7.45, and 8.02) were prepared fresh in house, described in the supplementary information, and measured with a standard benchtop pH meter. Phosphate buffered saline solution was also prepared in house and is described in the supplementary information. See supplemental information for complete component identification and preparation. 2.2. Adsorption procedure Approximately 10 mg of ND, ND-COOH, or ND-NH2 was added to 10 mL aqueous solutions of varied known concentrations of TET or VAN. The vials were closed with plastic threaded caps, sonicated in an ultrasonic bath until particles were well dispersed, and shaken at ambient temperature overnight to ensure equilibrium adsorption. The resulting mixture was centrifuged at 3500 rpm for 1 h to precipitate majority of ND particles. A 1 mL aliquot of the supernatant was taken and 0.125 mL of a 1 M KCl solution was added to it to salt-out any remaining ND particles from liquid phase (the small increase in volume due to the addition of KCl solution was properly taken into account when calculating drug concentration). This was centrifuged down at 3500 rpm for 45 min to ensure as much ND had been removed as possible before

Fig. 1. Structures of tetracycline (a, in neutral and zwitter ion forms) and vancomycin (b, in protonated form). The highlighted protonated amine groups (in green) on both drugs can interact with deprotonated carboxylic groups on surfaces of ND-COOH (c), and ND-NH2 (d). The highlighted hydroxyl groups on both drugs can interact with amine groups on ND-NH2. Known pKa are provided from literature (Ref. [6] for tetracycline and Ref. [5] for vancomycin). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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absorbance measurements. The peak absorbance of non-adsorbed VAN in solution was measured to be 280 nm, and that of nonadsorbed TET at 360 nm (Supporting Information, Fig. S1). A TECAN Infinite M200 with NanoQuant software 96 well plate reader was used to perform absorbance measurements.

bent, and Ceq is concentration of the adsorbtive in the environment in equilibrium with the adsorbent. The Freundlich isotherm [23] is an empirical model which describes the quantitative relationship between the adsorption and equilibrium concentration of the adsorptive:

2.3. Release procedure

ð1=nÞ A ¼ K F C eq ;

Drug loaded ND samples of the same loading extent were prepared as described in the Adsorption Procedure above. Supernatant in equilibrium with ND was carefully separated from drug loaded NDs by centrifugation at 3500 rpm for 1 h (without KCl addition) and pipetted out, and the remaining solid samples were dried at 85 °C overnight. 2 mL of a buffer solution (pH 4.09, 7.45, 8.02 or PBS) was added to the nanodiamond. The pellet in solution was then vortexed until the ND was re-dispersed. This solution was shaken for 24 h. After centrifugation at 3500 rpm for 1 h, 1.5 mL of supernatant was removed and 1.5 mL of fresh buffer of the appropriate pH added, the solution vortexed again for each time period. From the 1.5 mL aliquot of supernatant, 1 mL was taken and 0.125 mL of 1 M KCl added (the increase in volume due to the addition of KCl solution was properly taken into account when calculating drug concentration). One more centrifugation was done for 30 min at 3500 rpm. The resulting supernatant with no ND present was used to measure the concentration of the antibiotic in solution. Separate absorbance calibration curves of TET in pH buffers of 4.09, 7.45, 8.02 and PBS were recorded using the TECAN (for curves see supplemental information). The release profile of drug is plotted as a cumulative concentration over time. Each point on the release profiles was measured 3 times to calculate the average and standard deviation. To measure the concentration of VAN released by different pH solutions, a Pierce BCA protein assay was conducted. The working reagent (WR) for the assay was prepared according to instructions [21]. The WR was incubated with the drug solution in a ratio of 10:1 at 60 °C for 30 min, and read with the TECAN 96 well plate reader at 562 nm. 2.4. FTIR Fourier Transformed Infrared (FTIR) Spectroscopy was performed on samples of nanodiamond, tetracycline, vancomycin, and ND loaded with the drugs. Absorbance FTIR spectra were recorded on a diamond ATR Perkin–Elmer Spectrum One FourierTransform Infrared Spectrometer. A background spectra of air was collected and subtracted from the spectra of the samples recorded with a spectral resolution of 4 cm
1. A standard ATR correction provided in the Perkin–Elmer software (PE Spectrum v10.03.09) was applied to correct for relative band intensities. 3. Results and discussion In order to gain insight into the binding of the drugs to the surface, the experimental adsorption isotherms were fit by two commonly used models. The Langmuir isotherm (Eq. (1)) [22] assumes a monolayer adsorption by a finite number of localized identical sites on the surface of the adsorbent. Any possible interactions, favorable or unfavorable (including steric hindrance) between the adsorbed molecules, are excluded.

A ¼ Amax

K L C eq ; 1 þ K L C eq

ð1Þ

where A is adsorption capacity (mass of adsorbate per mass of adsorbent), Amax is maximal adsorption capacity of monolayer (mass of adsorbate per mass of adsorbent), KL is a constant which characterizes the strength of binding of the adsorbate to the adsor-

ð2Þ

where KF and n are empirical constants. In this model, there isn’t a restriction to the formation of layers of the adsorbate beyond a monolayer and it can be used to describe adsorption on heterogeneous surfaces with non-uniform population of adsorption sites. In the Freundlich isotherm, the value of adsorption is a sum over all adsorption sites, each with different affinity, where the sites of stronger affinity are occupied first followed by the sites of weaker affinity, and so on, corresponding to the exponential decay in the energy of adsorption. While there are a wide variety of isotherm models for adsorption, the Langmuir and Freundlich isotherms are commonly used for extracting relevant adsorption properties and thus suited our present goals. More sophisticated models, allowing to extract even more details about adsorption, can be used on later stages, when more experimental information on adsorption/desorption of different drugs on NDs with different surface chemistries become available.

3.1. Tetracycline The experimental adsorption isotherms of TET on ND, NDCOOH, and ND-NH2 are provided in Fig. 2a–c, respectively. Data fits by the Langmuir and Freundlich models are shown as curves overlaid with the measured data points. Based on correlation coefficients (Table 1), the adsorption data of TET demonstrate good agreement with the Langmuir and Freundlich models in the range of concentrations studied for all three nanodiamonds (as-rec ND, ND-COOH, and ND-NH2). Agglomerates were seen to form within the first hours of drug exposure for all ND drug combinations and all drug concentrations. This required additional vortex and hand agitation to break some of the larger agglomerates, which was repeated several times in addition to normal shaking during the equilibration period of 24 h. TET is known to form a zwitterion between the alpha amide and the corresponding hydroxyl group (shown in Fig. 1a) [6,24,25]. The zwitterion is known to dominate in aqueous conditions forming the hexahydrate complex from the TET HCl salt [26]. Monolayer capacities Amax calculated according to the Langmuir model reveal that the surface chemistry of NDs plays a role in the adsorption of TET. The as-received nanodiamond has the highest monolayer capacity at 132 mg/g. This can be attributed to the porous and inhomogeneous surface of the ND due to the presence of non-diamond amorphous carbon in this material [19]. The as-received ND also has the lowest binding constant for TET out of all the NDs. Without a strong attraction of specific functionality, the TET is not well bound. ND-COOH has the strongest binding constant due to the deprotonated carboxylic acid groups on the surface of the ND and the protonated alpha amid and tertiary amine group in the TET structure (Fig. 1a). The TET binding constant to ND-COOH is almost equal to ND-NH2 (both given in Table 1). Since the surface of the ND-NH2 is aminated to approximately 20 at.% [20] there are still residual carboxyl groups to bind the protonated TET giving a strong binding constant. Also, the deprotonated carboxyl group on TET can potentially bind with the aminated surface of the ND-NH2 however the reduced Amax suggests that the protonated amines on the surface of the ND are repelling the protonated zwitterion of TET. The strength of binding (KL in eq (1)) also varies in a wide range depending on the ND surface termination. The separation factor, or

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a

150

b

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A, (mg/g)

A, (mg/g)

75 100 75 50

50

25 25 0

0 0.0

0.2

0.4

0.6

0.0

0.8

0.2

Ceq, (mg/mL) 100

0.4

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c

d

1.0

ND ND-COOH ND-NH2

80 0.8 60 0.6

RL

A, (mg/g)

0.8

Ceq, (mg/mL)

40

0.4 20 0.2 0

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0.3

0.4

0.0

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0.3

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C0, (mg/mL)

Ceq, (mg/mL)

Fig. 2. Tetracycline adsorption data (points) on ND (a), ND-COOH (b), and ND-NH2 (c) nanodiamonds. Data fitted with non-linear least squares (NLLS) using Langmuir (blue solid lines) and Freundlich (red dashed lines) models. Equilibrium parameter RL as a function of initial concentration of adsorbtive in solution, C0, for tetracycline adsorbed by NDs with different surface chemistries (d). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 1 Best fit parameters of Langmuir and Freundlich isotherms for adsorption of tetracycline on NDs with different surface chemistries.

ND ND-COOH ND-NH2

Langmuir

Freundlich 2

Amax (mg/g)

KL (mL/mg)

R

KF

n

R2

132 86.4 76.1

16.4 21.9 20.1

0.86 0.86 0.89

10.9 11.8 5.92

2.49 3.16 2.23

0.88 0.96 0.88

equilibrium parameter, RL, can be calculated from the Langmuir isotherm as:

RL ¼

1 ; 1 þ K LC0

3.2. Vancomycin The adsorption isotherms of vancomycin on ND, ND-COOH, and ND-NH2 are provided in Fig. 4a–c, respectively. As before, the Langmuir and Freundlich models (lines) are overlaid with the measured data (points). In Table 2, the adsorption data of VAN demonstrates an overall better agreement with the Langmuir model in the range

ð3Þ

where C0 is the adsorbtive initial concentration. This parameter is related to the shape of the isotherm and can be used to define the adsorption process as irreversible when RL = 0, favorable when 0 < RL < 1, linear when RL = 1, and unfavorable when RL > 1 [27]. The equilibrium parameter for the TET isotherms is given in Fig. 2d. The values come very close to 1 at low concentrations (much below 0.005 mg/mL) indicating an irreversible adsorption. Since these concentrations are too low for TET to be effective in a human body, the full range of the equilibrium parameter over the concentration range is favorable for a reversible adsorption. The equilibrium parameter comes close to 0 above 0.5 mg/mL where the adsorption starts to become unfavorable as also noted by increased scatter in the adsorption isotherms for Ceq > 0.3 mg/mL. FTIR spectra of ND-COOH, TET, and the ND-COOH loaded with TET are presented in Fig. 3. FTIR spectroscopy was done to determine if TET was bound to the ND-COOH. The CAN bond from TET is observed at 1453 cm
1 [6]. A relative decrease in the

C-O 1625 C=O 1760

Overlap N-H/O-H 3400

C-N 1453

ND-COOH ads TET

Absorbance (a.u.)

Nanodiamond

intensity of C@O at 1760 cm
1 is observed after the adsorption of tetracycline. This can be due to the interaction of the amine groups of TET with the carboxylic acid groups of ND-COOH.

ND-COOH

TET

500

1000

1500

2000

2500

3000

3500

4000

Wavenumber (cm-1) Fig. 3. FTIR spectra of tetracycline, oxidized nanodiamond, and a sample of NDCOOH loaded with TET. Dashed lines indicate primary functional groups.

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a

b

125

200

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75

A, (mg/g)

A, (mg/g)

250

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50 25

50

0

0 0.0

0.2

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120

c

100

60

RL

A, (mg/g)

80

40 20 0 0.0

0.2

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0.6

Ceq, (mg/mL)

Ceq, (mg/mL)

0.6

0.8

1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

d

ND ND-COOH ND-NH2 0.0

0.1

0.2

0.3

0.4

0.5

C0, mg/mL

Ceq, (mg/mL)

Fig. 4. Vancomycin adsorption data (points) on ND (a), ND-COOH (b), and ND-NH2 (c). Data fitted with NLLS using Langmuir (blue solid lines) and Freundlich (red dashed lines) models. Equilibrium parameter RL as a function of initial concentration of adsorbtive in solution, C0, for vancomycin adsorbed on different NDs (d). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

of concentrations studied for the as-received, ACOOH, and ANH2 terminated NDs. Monolayer capacities Amax calculated according to the Langmuir model, demonstrate again that the surface chemistry of NDs and amorphous carbon present in as-received ND play a significant role in adsorption. The as-received nanodiamond has the highest monolayer capacity at 288.67 mg/g. As-received NDs have slightly higher correlation coefficient of Langmuir fit with VAN, as opposed to purified and chemically modified powders. However, the binding strength (KL) of VAN adsorbed on NDCOOH is twice as much due to the VAN becoming overall positively charged in solution from its two alkylamine groups and multiple amide nitrogens (positive), and single carboxylic acid group (negative) (Fig. 1b) [5,28]. However, it is difficult to choose which model fits the VAN isotherms on ND-NH2 better due to a very low binding strength of VAN to ND-NH2, manifested by the isotherms not leveling in Fig. 4c as opposed to Fig. 4a and b. Additionally, VAN does not seem to start to bind to ND-NH2 until a minimum equilibrium concentration of 0.08 mg/mL is achieved (manifested by the almost flat part of the isotherm in the range of C0 = 0–0.1 mg/mL in Fig. 4c). Up to this point, no VAN is seen to bind to the NDNH2 which suggests that in aqueous solution VAN is repelled by

Table 2 Best fit parameters of Langmuir and Freundlich isotherms for adsorption of VAN on NDs with different surface chemistries. Nanodiamond

ND ND-COOH ND-NH2

Langmuir

Freundlich

Amax (mg/g)

KL (mL/mg)

R2

KF

n

R2

288.67 125.81 183.95

5.56 12.12 1.45

0.94 0.89 0.92

7.00 9.46 0.77

1.758 2.427 1.344

0.88 0.81 0.89

the protonated amine groups on the ND-NH2. The equilibrium parameter for the VAN isotherms is given in Fig. 4d. The values are very high in the concentration range. The ND-NH2 curve is almost linear and suggests not only is the VAN reversibly binding, but indeed binding very weakly. This points again to the prevailing role of electrostatic interactions between the drug and ND: as soon as electrostatic interactions become unfavorable, even large molecules such as VAN, which have plenty of sites for interactions of other types with the surface, cannot stay attached to it. The negatively charged ND-COOH does work however and has both a good binding constant and monolayer capacity. FTIR spectra of as-received nanodiamond (ND), VAN, ND loaded with VAN, and ND with VAN after 8 rinsing cycles are shown in Fig. 5. All characteristic bands in ND are present and are identified specifically elsewhere [19,29]. It can be observed that the characteristic C@C stretch at 1495 cm
1, which is present in VAN [30], is also present in the loaded ND sample. In the VAN adsorbed sample that has been rinsed, there is still a shoulder absorbance of C@C present, but at a reduced intensity. This suggests that there may still be some amount of VAN present on the ND that is irreversibly attached. Likewise, a similar trend can be seen for the CAOAC bending at 1230 cm
1. What is noteworthy is that the CAH stretching of the ND at 2950 cm
1 is substantially reduced after the loading of VAN. One explanation is that some water soluble contaminants containing CAH bonds (oxidized with hydroxyl or other water soluble functional groups) on the surface of as-received ND are removed during the loading process and remain in the supernatant after centrifugation. The washing off of these CAH containing species may be facilitated by the presence of VAN, which in this case may compete with these species for ND adsorption sites and/or act as a surfactant, stabilizing these species in solution. It also explains why the

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Adsorbance (a.u.)

C-O-C 1230 C=C 1495

C=O 1760

ND ads VAN post 8 rinses

C-H 2950

Overlap N-H/O-H 3400

ND ads VAN

ND

VAN 500

1000

1500

2000

2500

3000

3500

4000

Wavenumber(cm-1) Fig. 5. FTIR spectra of vancomycin, as-received ND, and a sample of ND loaded with VAN immediately after loading and after 8 rinsing cycles. Dashed lines are primary functional groups.

CAH stretch bands do not return after rinsing VAN from the ND (Fig. 5). Regardless of the mechanism, the concentration of these species in solution is low and does not interfere with UV–Vis analysis. 3.3. pH triggered release The controlled release of tetracycline, as with many other drugs attached largely with electrostatic interactions, can be accomplished by changing the pH of the environment [31–33]. The release of TET from ND, ND-COOH and ND-NH2 in buffer solutions of pH 4.09, 7.45, 8.02 and PBS (pH 7.4) is presented in Fig. 6a–c respectively. The concentration of TET was determined from UV–Vis absorbance. TET, which has a primarily positive charge in neutral aqueous environment from its amine groups, will bond with ND-COOH, which has a strong negative charge when deprotonation occurs. In acidic pH conditions, when the attractive electrostatic interactions are suppressed due to association of H+ and COO
, one would expect some TET to be released from the surface of ND-COOH. This is the observed effect in the case of pH 4.09 for all nanodiamond types. The porous surface of as-received ND is capable of holding more drug but it is mainly held due to weak non-specific interactions. From the release profile in Fig. 6a, no more than 11% of TET is released from the surface of as-received ND even at the most favorable pH 4.09. The small release and its insensitivity to pH most likely indicate that the drug is trapped in the pores of amorphous carbon present in as-received ND. The enhanced release of TET from ND-COOH in an acidic environment indicates that ND-COOH is not a good choice for a long term slow release of TET when pH is <7. In contrast, PBS (pH 7.4) keeps the release of TET from ND-COOH to approximately 7% and the lowest of all buffer solutions used. The results show that PBS buffer overall reduces the release of TET from ND and ND-COOH by 40–50%. This is likely due to a higher ionic strength of PBS (which represents electrolyte composition of blood plasma) as compared to other buffers, which may result in reduced solubility of TET, as well as dispersion stability of ND and ND-COOH in water – both will slow down the release. The release of TET from ND-NH2, for neutral to basic pH tested, is the lowest (most fall below 2%). However, a change to an acidic pH triggers the release of adsorbed TET from ND-NH2 (Fig. 6c, squares) though it still remains below

10% cumulative release. The binding of TET to ND-NH2 is a complex one. While ND-NH2 has the lowest Amax with TET compared to as-received ND and ND-COOH, it has an equal binding constant with TET compared to ND-COOH. As depicted in Fig. 1a, there are COO
and NH+3 on the surface of ND-NH2 which can complex with the zwitterion form of TET. The fact that more TET is released in acidic conditions suggests that the COO
on the surface of the ND-NH2 plays the larger role in adsorption. The leaking of drugs in the body is of particular concern for VAN due to the drug’s ototoxicity and nephrotoxicity [3,34,35]. Thus control over the release of VAN is even more desirable than TET. To determine the release of VAN as a function of pH a BCA assay was used. The BCA assay is a very sensitive and reliable method for determining the concentration of polypeptides in solution when the concentration can potentially be very low [36]. VAN was used to build a calibration curve for the BCA assay. The maximum release of vancomycin from as-received ND for pH 4.09, 7.35, 8.02 and in PBS was 58%, 72%, 72%, and 52% respectively over nine days (Fig. 6d). The high initial release of VAN from NDs with all three surface chemistries within the first day (above 20% for all pH) supports the low binding strength measured in the adsorption study (Table 2). Following the first day all samples showed steady release. The lowest release of VAN is observed in the acidic environment. Results show greater VAN release in a basic environment which indicates that the positive charges on the vancomycin amine groups (Fig. 1b) and any negative charged groups on the ND should play a crucial role in the adsorption. An alkaline environment reduces the positive charge of the amine groups of VAN and, as a result, it desorbs more readily from the ND. Same as for TET, PBS greatly reduces the release of VAN (compare the curves for a buffer with pH 7.45 and PBS with pH 7.4), although in this case the cumulative release reaches much higher values even in PBS – 52% over the nine day period. Understanding the monolayer capacity and strength of binding provides the basis of knowledge on the effects of ND chemistry on drug adsorption, justifying the need to develop a library that can be used as a reference for building ND drug delivery systems. Furthermore, with the addition of the controlled desorption information, a significantly more specialized and precise system can be made. In our previous report [18], a trend was observed with doxorubicin (DOX) and polymyxin B (PMB) on the different NDs. Namely, that even though there was a high monolayer capacity for doxorubicin with ND-NH2 there was a low binding strength compared to other ND–drug pairs. This trend is extended here with the addition of tetracycline and vancomycin (Fig. 7). The strength of binding is considerably lower for these two antibiotics, but relatively high monolayer capacities are observed. It is also indicative by the low binding constant of vancomycin that the structure and thus conformation that the molecule takes on the surface of the ND plays a role in the binding strength. When one compares doxorubicin with tetracycline, they both have polycyclic structures. However, the close proximity of oppositely charged functional groups on TET (Fig. 1a) most likely hinders a stronger electrostatic interaction with the adsorbent. The release of TET would indicate that its binding on all NDs is sufficiently strong, as no more than 20% is released even at an acidic pH (Fig. 6a–c). This gives promising evidence that TET would likely remain attached to the ND before reaching its target. Vancomycin on the other hand binds the weakest and it is most likely due to its bulky ring structure, which is stiffer than some other linear polypeptides. Other studies have attached VAN by multiple targeted cites, used covalent bonding or encapsulation in polymer nanoparticles [37,38]. This strengthens the net interaction between the VAN molecule and the delivery nanoparticle. As can be seen with

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14

25

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Cumulative Release %

12 10 8 6 4 2 0

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0

2

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40

20

0 0

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Time (days)

Fig. 6. Tetracycline controlled release from as-rec ND (a), ND-COOH (b), ND-NH2 (c) and vancomycin controlled release from as-received ND (d) with varying pH of 4.09 (-j-), 7.45 (-d-), 8.02 (-▲-), and also with PBS (pH 7.4) (-r-). The error bars represent standard deviation of each point. For the vancomycin release profile, after the first day the deviation is around ±0.5% cumulative release.

Fig. 7. KL vs Amax from Langmuir model for doxorubicin, polymyxin B, tetracycline, and vancomycin adsorbed on NDs with different surface chemistries (data from Ref. [18] and Tables 1 and 2).

the release study presented here, 50–70% of VAN can be desorbed from the surface of the NDs (Fig. 6d). ND-COOH had a slightly stronger binding with a lower adsorption monolayer capacity. To minimize drug leaks and adverse reactions in the body, this particular delivery system would be most promising for vancomycin. This information can contribute to the rational design of the ND based drug delivery systems for different purposes exemplified by a situation when a long term sustained release is required, in contrast to a situation when a sharp increase of the drug concentration has to be achieved over a short time.

4. Conclusions The adsorption results show that tetracycline binds strongly to the surface of ND, and ND-COOH. Vancomycin binds the weakest of all the drugs tested at isothermal equilibrium in this and previous studies. The differences can be explained by steric interferences and conformations of the drug on the surface of the ND. Both TET and VAN show a reduced Amax and low binding strength for ND-NH2, respectively. For VAN, this can be attributed to the number of amide and amine groups which can be protonated; however

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future studies of conformational changes to VAN when adsorbed would elucidate the mechanism of adsorption. For TET, it is likely that the reduced Amax is due to the protonated amines on the surface of the ND-NH2 repelling the protonated TET, however the KL on ND-NH2 is equally as large as on ND-COOH. Since the surface of the ND-NH2 is aminated to approximately 20%, it can be assumed that the TET binds to residual carboxylic acid groups. Furthermore, TET releases from ND-NH2 far less in basic pH than in acidic pH which is the same trend in ND-COOH. The release profile for TET on as-received ND indicates that in this case the binding is not specifically controlled by electrostatic interactions. If it were, a higher release, as can be seen with ND-COOH and ND-NH2 with acidic pH, would be expected. Loading TET or any drug under set pH conditions may potentially increase loading capacity. Indeed, studying the conformational changes of the drugs on the ND surfaces is warranted to fully understand what influences Amax and KL. Additionally, release studies should be extended to assess how long the drugs are stable on the NDs. Using PBS in the release studies was critically important for future physiological studies. This work further contributes to a rational design of ND-enabled drug delivery platforms. Because the adsorption work in this article focused on two isotherm models, future work will need to focus on fully understanding the mechanism of adsorption which can only be accomplished by studying the adsorption of many more drugs with different isotherm models. These experimental results contribute to the rational design of ND enabled theranostic platforms [11,39,40]. Further steps in this direction will require detailed experimental and theoretical analysis of the effects of ND surface chemistry in the mechanisms governing the adsorption and release of drugs. Furthermore, the agglomeration state of the NDs with each drug has been shown to play an important role in the effectiveness of drug delivery as well as matrix and interface effects [41,42]. These will have to be studied for many drug nanodiamond surface pairs to create a detailed road map to using nanodiamonds as an effective theranostic drug delivery platform. This work demonstrates that the surface chemistry plays an important role in the binding affinity of drugs which will influence how they can be adsorbed, transported in the body, and released. Moreover, the declining antibiotic potency necessitates systemic use of antibiotic cocktails in higher concentrations, which have harmful effects to the human body and contribute to development of resistance of bacteria to these drugs. By delivering clinically approved antibiotics with nanodiamond and their release locally, it may be possible to restore their potency while minimizing negative side effects and development of bacterial resistance. Acknowledgements Special thanks go to Dolores Conover, Dr. Kara Spiller and the CORE facility of the BIOMED Department of Drexel for use of their lab space and use of the TECAN, and to Dr. Jon Soffer and the Chemistry Department of Drexel for use of their FTIR. This work was supported by Drexel University through Drexel-SARI Center. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcis.2016.01.062. References [1] C. Demetzos, N. Pippa, Advanced drug delivery nanosystems (aDDnSs): a minireview, Drug Deliv. 21 (2014) 250–257. [2] R.A. Al-Bayati, A.S. Ahmed, Adsorption–desorption of trimethoprim antibiotic drug from aqueous solution by two different natural occurring adsorbents, Int. J. Chem. 3 (2011) 21–30.

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