Carbon-Dot-Coated Alginate Beads as a Smart Stimuli-Responsive

Letter www.acsami.org

Carbon-Dot-Coated Alginate Beads as a Smart Stimuli-Responsive Drug Delivery System Sristi Majumdar,† Gargee Krishnatreya,† Neelam Gogoi,† Debajit Thakur,‡ and Devasish Chowdhury*,† †

Material Nanochemistry Laboratory, Physical Sciences Division and ‡Life Sciences Division, Institute of Advanced Study in Science and Technology, Paschim Boragaon, Garchuk, Guwahati 781035, India S Supporting Information *

ABSTRACT: In this work, we report a smart stimuli-responsive drug delivery system (DDS) that can release drug depending upon the amount of pathogen (MRSA) present in the target. A greater amount of MRSA in the system will lead to more release of drug and vice versa. Carbon-dot-coated novel alginate beads (CA-CD) exhibiting superior stability was successfully used as smart drug delivery vehicle. Garlic extract (GE), which contains allicin, was taken as model drug system to demonstrate the phenomena. It was observed that GE loading was 19 and 78% with CA and CA-CD, respectively. CA-CD-GE shows pHdependent controlled drug release, which results in increased therapeutic efficiency. CA-CDGE is not only stimuli responsive but also a controlled drug release system as it releases drug according to the pathogen concentration (MRSA). All the three factors viz. drug release, MRSA concentration and pH of the medium are interdependent as when the cell divides, it produces secondary metabolites that lead to the decrease in pH of the medium. The drop in the pH value triggers drug release from the beads. And the effect of the drug is reflected by the MRSA cell death. Hence, we demonstrate a smart stimuli responsive DDS. However, such DDS will be useful in cases where increased amount of pathogen in the system will lead to reduction in pH. KEYWORDS: carbon dot, drug delivery system, alginate, stimuli responsive, MRSA

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of cholesterol.14 The system shows enhanced PL intensity upon addition of cholesterol. As a promising nanomaterial, carbon dots are broadly used in different targeted drug delivery system (DDS). There are very few reports of use of CDs as DDS. Wang et al. used fluorescent carbon dots prepared from bovine serum albumin as pHcontrolled release for doxorubicin (DOX).15 Similarly, Lai et al. used CDs from glycerol and prepared doxorubicin (DOX) loaded CDs@mSiO2−PEG nanocomposites and showed controlled release of DOX.16 Tang et al. showed direct and sensitive Förster resonance energy transfer (FRET)-based CDs drug delivery system (FRET-CDs-DDS) via a direct surface coupling strategy, which faciliates real-time monitoring of drug release.17 Pandey et al. demonstrated carbon-dot- functionalized gold nanorod mediated delivery DOX.18 DDS are designed so as to deliver sufficient amount of drug to the target area so as to maximize therapeutic reaction and to minimize collateral damage. Stimuli-responsive, controlled-release DDSs are designed to entrap drug until some change is triggered in the system to release the required amount of drug. Most of the DDS are designed to release a predetermined amount of drug and are not according to what is required at the target. In this work we have tried to develop a pH responsive DDS system of carbon-dot-coated alginate beads (CA-CD) which can release

or many years, quantum dots (QDs), especially II−VI semiconductor QDs, have been used in the biomedical sector because of their tunable fluorescence property.1 But its highly toxic trait led to the urgent need for an advanced nano material that can replace these QDs.1,2 Amidst this, carbon dots (CDs)3 were introduced as the newest class of carbonic nanomaterials with dimension less than 10 nm. The unparalleled properties of CDs have inspired extensive study on them and resulted in many unprecedented findings. CDs have always been well-touted as a potential class of biomaterials by virtue of their low toxicity, biocompatibility and easy synthesis.4−8 Also, the complex cellular micro environment exerts significant challenges in real time drug delivery monitoring in physiological systems. The tunable fluorescence properties of CDs make them the excellent candidate for bioimaging.9,10 In addition to this CDs are also excellent electron donor and electron acceptor, which makes them highly applicable for detection of various biological entities. In 2014, Cui et. al, designed a highly sensitive mercury sensor based on CD-labeled oligodeoxyribonucleotide which has a detection limit of 2.6 nM.11 Following this, in the same year, Dai et al., developed a FRET system with an amino acid functionalized CDs and Au nanoparticle.12 This system was successfully used to detect organic adulterates like melamine in milk samples. In 2015, Liu et. al, developed a CD-based biosensor for the sensitive and selective detection of hyaluronidase which plays a pivotal role in cancer malignancies.13 Recently, Bui et al. successfully used a CD-hemoglobin complex for the detection © XXXX American Chemical Society

Received: August 30, 2016 Accepted: December 1, 2016

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DOI: 10.1021/acsami.6b10914 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Scheme 1. Schematic Representation of the Protocol Adopted of Preparation of Carbon-Dot-Coated Alginate Beads and Subsequent Loading of Allicin (Garlic Extract)

the drug depending upon the amount of pathogen (MRSA) present in the target. Alginate being one of the most biocompatible materials has structural similarity with the extracellular matrices of living organism. Derived from brown seaweed, they are praised for their low toxicity, biodegradability, and relatively low cost. But most importantly they have properties like mechanical stiffness, cell attachment ability and binding or releasing the bioactive molecules upon certain modifications, which makes them a valued material for many biomedical applications.19−21 Our laboratory has already developed carbon-dot-coated alginate beads with pH-responsive drug delivery.19 We went one step further and used CA-CDs, taking garlic extract (GE) that contains allicin as model drug system to demonstrate the proof of concept of smart pH-responsive DDS. We realized that pH of the medium depended on the amount of MRSA cells present. Excessive growth of MRSA cell results in a decrease in the pH of the medium. As the pH of the system decreases CACDs system releases the drug. If the cell growth was ceased, the pH of the medium remains stagnant and hence no further drug release was observed. Thus, the evolution of pH of the medium can be used as the index for the MRSA cell growth progression. We have used the pH depended growth to develop a smart pH responsive DDS. The prepared CA-CD beads are used as a smart drug delivery system (DDS) in order to overcome the drawbacks of conventional DDS. Carbon dots were prepared and fully characterized as per earlier established protocol. The characterization of carbon dots is presented in detail in Figure S1. CACD beads were prepared by keeping the beads soaked in the CD solution for 6 h and then rinsed properly with distilled water to remove any unbound CDs. Drug loading was carried out as shown schematically in Scheme 1. For maximum drug loading, the beads were soaked in 0.1% GE solution and kept undisturbed for 4 h. The beads were properly washed with distilled water and soaked in tissue paper. The beads were then air-dried to an extent where there is no further loss of weight. The beads were weighed again to calculate the % of drug loading.

The following formula was used to determine the drug loading into the beads: The percentage drug loading by CA-CD beads was found to be 77.98%, a value much higher than the percentage drug loading by CA beads which was only 19.37%.This proved that functionalization of the beads with CDs has promoted drugpolymer interaction resulting in increased drug encapsulation by several folds.UV−visible spectroscopy was carried out on CA, CA-CD and garlic extract loaded carbon dot-coated calcium alginate beads (CA-CD-GE). Figure 1 shows the UV−

Figure 1. UV Spectra of CA, CA-CD, and CA-CD-GE.

visible spectra of CA, CA-CD and CA-CD-GE beads. The spectra represents the typical absorption band of a polysaccharide exhibiting peaks at 211 nm in CA, 208 and 247 nm in CA-CD, and 212, 233, 264 nm in CA-CD-GE, which might be due to π → π* and n → π* type of electronic transitions occurring in them on excitation. A shift in 211 nm peak of CA to 208 nm on coating of CA with CDs in CA-CD suggests the successful interaction of CDs with alginate moieties. After B


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evident from photographic image.To determine any morphological changes in the beads due to the incorporation of CDs and GE, we carried out scanning electron microscopy (SEM) on the beads. Figure 3 represents the SEM images of the beads.

loading GE into CA-CD, visible shifts in the peaks of CA-CD indicate efficient loading of drug with occurrence of a shoulder peak at 264 nm, which could be due to nonbonding electrons of S−SO present in allicin of GE. To have more information regarding the interaction between GE, CA, CA-CD, and CA-CD-GE, FTIR analysis was done and presented in Figure 2a. The most abundant compound of GE is

Figure 3. Representative SEM images of (a) CA, (b) CA-CD, and (c) CA-CD-GE beads.

Figure 3a shows the enhanced micro structure of CA beads with unsheared surface and very low porosity. It is observed from SEM micrograph that coating of CA with CDs results in increase in the porosity of the material. Moreover, CDs are seen uniformly distributed on the surface of beads. Loading of GE into the CA-CD bead resulted into the tranquil surface of the beads. Drug release study by CA-CD-GE at different pH: The functionalized hydrogel was designed to release drug at low pH, and hence, the release profile was investigated by studying the pH responsive behavior of CA-CD-GE beads in different pH mediums. CA-CD-GE beads were kept in different pH solutions for 24 h. pH-responsive drug release of the beads was studied in four different pH solutions (viz. pH 3, pH 5, pH 7, and pH 9). Aliquots of 3 mL were withdrawn from the release medium periodically and added back after each sampling to maintain constant volume of the release medium. The amount of GE release from the beads was determined by measuring the UV−visible absorption in the range of 190−300 nm for each sampling. Figure 4 shows the UV−vis spectra of the drug release study conducted at different pH. It was observed that the beads were stable in acidic pH but disintegrates in basic pH. At pH 5, maximum swelling of the beads were observed by naked eyes and also maximum drug release was noted by UV−visible study, whereas at pH 3 and pH 7, the beads showed considerably less swelling and at pH 9, the beads were unstable and became completely dispersed. The pH-dependent release of the drug might be due to the result of different chemical interaction between the drug molecules and the CA-CD. When pH ranges from 5 to 7, protonation of COO− groups is less which results in weaker Hbonding between COO− and COOH by mutual charge repulsion of COO−. Also, the mobile ions inside the beads cause high osmotic pressure which in turn results in loosening of network structure and hence swelling is observed. In basic medium, due to the presence of Na+ ions an ion-exchange

Figure 2. (a) Stacked FTIR spectra of 0.1% GE, CA, CA-CD, and CACD-GE; (b) photographs of beads (i) CA, (ii) CA-CD, and (iii) CACD-GE.

allicin which contain SO and S−S groups. The GE spectrum shows peak at 525 and 1039 cm−1 for S−S and SO, respectively. Thus, the structure of allicin was determined by FTIR spectra.22 CA spectrum shows peaks at 3350 cm−1 (symmetric and asymmetric stretches of OH group), 2127 cm−1 (CC vibrational stretch), 1635 cm−1 (N−H vibrational bend), 1417 cm−1 (C−C vibrational stretch in ring), and 1027 cm−1 (C−N vibrational stretch). Coating of CA beads with CD resulted in the generation of new troughs throughout the spectrum. C−H stretch appears as small bands at 2919 and 2851 cm−1. The C−H vibrational stretch gives evidence for the presence of chitosan moiety present in CDs. The N−H band has shifted from 1635 to 1598 cm−1 which might be due to the electrostatic interaction between − NH3+of chitosan CD and −COO− group of CA. Appearance of a band 1298 cm−1 depicts the presence of C−O vibrational stretch which confirms the presence of chitosan moiety. Loading of GE into the CA-CD beads resulted in the formation of a peak at 1032 cm−1 that is due to the SO vibration. This confirms the presence of allicin, the active compounds of GE in the bead. The photographic image of typical CA, CA-CD and CA-CDGE beads are shown in Figure 2b. The transition of the wrinkled to spherical bead can be observed with bare eyes as C


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in experimental section. 15 mL of Nutrient Broth (NB) was prepared in 4 culture tubes and autoclaved at 121 °C and 15 lb pressure for 20 min. The forthcoming work was carried out inside laminar air flow to maintain the sterile condition. One of the tubes was kept aside for further use in the instrumental analysis for baseline correction. Remaining three tubes were labeled as “control”, “CA-CD” and “CA-CD-GE”. The control tube contained NB and MRSA to monitor the undisturbed growth of MRSA. The second tube named CA-CD contained NB, MRSA and CA-CD bead (without drug) to determine any superfluous effect in the system. The third tube contained NB, MRSA and drug loaded beads hence named “CA-CD-GE”. The second and third tube contained 10 beads of almost same size. All the tubes were then incubated at 37 °C at 175 rpm 500 μL of sample was taken out from each test tube and the optical density was measured in UV−vis spectrophotometer at 600 nm at different time interval. The OD of each culture tube was measured at zeroth, first, second, fourth, fifth, sixth, seventh, eighth, 10th, 12th, 18th, 20th, 21st, 22nd, and 24th hour. Figure 5a shows the pH dependent drug release by beads at different

Figure 4. UV analysis of drug release by CA-CD-GE beads at different pH.

reaction occurs between Na+and Ca+. Thus, the network gets disrupted in aqueous NaOH solution resulting in dispersion of beads.19 An FTIR spectrum of the CA-CD-GE bead was recorded in order to determine the pH dependent drug release profile of the beads. The result is discussed in Figure S3. The main aim of this study was to develop an efficient, effective, and smart in vitro drug delivery system that can release drug depending upon the concentration of pathogens (MRSA) in the target. GE show excellent activity against MRSA (detail in Figure S2. The MIC/MBC of allicin to be effective against MRSA was also determined using agar diffusion method and is discussed in detail in the Supporting Information. Scheme 2 illustrates the procedure adopted to show a smart drug delivery system. MRSA strain and CA-CD-GE beads were cocultured and their UV absorbance was measured from after a definite interval of time. The detailed method used to evaluate drug release by CA-CD-GE beads has been described in detail

Figure 5. (a) UV analysis of cell death at different time intervals; (b) conc. of cell vs cell death assay at different time intervals.

time interval. From the figure, it can be inferred that the MRSA cells continued to divide and increase in cell mass. Similar growth pattern was observed in the control and CA-CD tubes unlike the CA-CD-GE tube where after 10th hour the O.D. of

Scheme 2. Schematic Representation of Protocol Adopted to Study the in Vitro Drug Release by CA-CD-GE Beads

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the culture started to decrease significantly. This might be due to the excessive growth of MRSA which resulted in the production of various secondary metabolites. These secondary metabolites in turn resulted in the decreased pH of the medium. As our system was designed to release drug at low pH, hence the CA-CD-GE beads released the drug resulting in cell death. Here, we would like to mention that we also carried out a blank experiment to determine the therapeutic efficiency of CA-GE and CD-GE. The bare CA beads are unstable in GE solution and get degraded within 30−45 min in the GE solution, whereas the CD-GE solution does not show any pHdependent cell death as the drug is not encapsulated within any system and are freely available in the medium. For the substantiation of our claim, another study was carried out where the concentration of MRSA cell was varied. In this study three different culture tubes were used which contained NB, 10 equal sized CA-CD-GE beads and different concentration of MRSA cells. The first, second and third tubes carried 4 × 108 cfu/mL, 8 × 108 cfu/mL, and 1.2 × 109 cfu/mL, respectively. All the three tubes were incubated in optimal growth conditions. 500 μL aliquot from all three tubes were taken in three different cuvettes and the O.D. was measured at 600 nm. This was carried out for zeroth, second, fourth, sixth, eighth, 10th, 12th, 18th, 20th, 22nd, and 24th hour. Figure 5b shows the histogram of the O.D. taken at different intervals. It was observed that the tube with the highest cell concentration, i.e., 1.2 × 109 cfu/mL, showed the highest cell death after 24 h. This demonstrates a very important observation of the study that as the cell concentration increases, more secondary metabolites are produced and the pH of the medium decreases. This results in the release of drug from CA-CD beads as it is strategically made to work only at low pH. In summary, we have fabricated a stimuli-responsive smart drug delivery system that releases drug depending upon the local environment in our case concentration of pathogen present. This system is highly responsive toward low pH and upon sensing the same, it shows controlled drug release that results in increased therapeutic efficiency. CA-CD was used as drug delivery system (DDS) and garlic extract (GE) which contains Allicin was taken as model drug to demonstrate the proof of the concept. This unique drug delivery system is not only stimuli-responsive but also is a controlled drug release system as it releases drug according to the cell concentration. All the three factors viz. drug release, cell concentration, and pH of the medium are interdependent as when the cell divides, it produces secondary metabolites which lead to the decline in pH of the medium. The drop in the pH value triggers drug release from the beads. And the effect of the drug is reflected by the MRSA cell death. Such smart drug delivery systems hold utmost importance in the near future as they can find multi applications in the biomedical field.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +91 361 2279909. Tel: +91 361 2912073. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

D.C. thanks SERB, New Delhi, Grant SB/S1/PC-69/2012, and BRNS, Mumbai, Grant 34/14/20/2014-BRNS, for funding. S.M thanks BRNS for a fellowship.

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ASSOCIATED CONTENT

S Supporting Information *

. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b10914. Experimental section (Materials & Methods), characterization of chitosan carbon dot, Allicin as an active compound in garlic extract, and pH-dependent drug release (PDF) E


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Carbon-Dot-Coated Alginate Beads as a Smart Stimuli-Responsive

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