Development of Gelatin Nanoparticle-Based Biodegradable

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Development of Gelatin Nanoparticle Based Biodegradable Phototheranostic Agents: Advanced System to Treat Infectious Diseases seema kirar, Neeraj Singh Thakur, Joydev K. Laha, Jayeeta Bhaumik, and Uttam Chand Banerjee ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.7b00751 • Publication Date (Web): 03 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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ACS Biomaterials Science & Engineering

Development of Gelatin Nanoparticle Based Biodegradable Phototheranostic Agents: Advanced System to Treat Infectious Diseases Seema Kirar 1, Neeraj S. Thakur 2, Joydev K. Laha 3, Jayeeta Bhaumik 1,# and Uttam C. Banerjee1, 2,*

1

Department of Biotechnology

2

Department of Pharmaceutical Technology (Biotechnology)

3

Department of Pharmaceutical Technology (Process Chemistry)

National Institute of Pharmaceutical Education and Research (NIPER) Sector-67, S.A.S. Nagar-160062, Punjab, India. #

Present address: Center of Innovative and Applied Bioprocessing (CIAB), Sector-81

(Knowledge City) S.A.S. Nagar-140306, Punjab, India. *Corresponding author E-mail: [email protected]

ACS Paragon Plus Environment

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

Abstract Rose Bengal (RB) conjugated and entrapped gelatin nanoparticle (GNPs) based biodegradable nanophototheranostic (Bd-NPT) agents have been developed for the efficient antimicrobial photodynamic therapy. The study reveals that the use of gelatin nanoparticles could bypass the chemicals such as potassium iodide, EDTA, calcium chloride and polymyxin nonapeptide for the penetration of drug into the cell membrane to achieve antimicrobial activity. We demonstrated that the singlet oxygen generated by the biodegradable gelatin nanoparticles (BdGNPs) could damage the microbial cell membrane and the cell dies. The key features of the successive development of this work include; the environmentally benign amidation of RB with GNPs which was so far unexplored and the entrapment of RB into the gelatin nanoparticles (GNP). The RB-GNP exhibited potent and broad-spectrum antimicrobial activity and could also be useful to treat multidrug-resistant microbial infections. Keywords: Nanophototheranostic agents, Singlet oxygen, Conjugation, Antimicrobial PDT Introduction Antimicrobial photodynamic therapy (aPDT) is a promising approach to the medication of superficial and localized infections.1 It is an alternative therapy for controlling the bacterial infections based on the use of the agents different from antibiotics.2 Photosensitizers (PSs) are indispensable chemical elements of photodynamic therapy (PDT), which upon irradiation by light of specific wavelength, are activated and generate singlet oxygen that causes tumor/bacterial cell demolition.3 These highly reactive species are able to attack most of the biomolecules such as proteins, nucleic acids and lipids.4 Most of the PSs under investigation for the treatment of microbial infections are based on xanthenes (rose bengal),4–7 phenothiazines (methylene blue and toluidine blue O),8–10 perylenequinones (hypericin),11 acridine,12 indocyanine green,13 malachite green14 and rhodamine.15 Among these PSs, rose


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bengal has been frequently studied for antimicrobial photodynamic inactivation because of its higher photo-stability, higher extinction coefficient, higher fluorescence quantum efficiency and less toxicity.1 It has little or no activity in mediating antimicrobial photodynamic therapy of Gram-negative bacteria, unless methods are employed to permeabilize the outer membrane, for instance by combination with EDTA,16 or polymyxin nonapeptide,17,18 calcium/magnesium,19 polymers,20 silica nanoparticles21 and increase the singlet oxygen generation using KI.4,22 It has been reported that the antimicrobial activity of rose bengal can be increased against Gram-negative or Gram-positive bacteria and fungal yeast using the chemicals which might be harmful to the normal cells. 4,16–23 The other important problem of RB is the rapid excretion from the body and relatively uncontrolled release of the drug due to hydrophilicity.24 Nanotechnology is a promising strategy for overcoming these drawbacks and to develop an appropriate drug delivery system.23 Unlike traditional use of RB, RB with gelatin,

obviates

the

requirement

of

chemicals,

thereby

empowering

superior

biocompatibility, sustainability and control drug release. Moreover, RB-GNP conjugates have proven to be a powerful variant of traditional couplings enabling rapid activity of RB. No reports are available on the development of any nanotheranostic agent on aPDT via RB conjugated gelatin. Here, a new aPDT agent was designed using a promising biomaterial candidate gelatin which is non-toxic, cost-effective, non-immunogenic and biodegradable in nature.25 Gelatin is taken by the people as food which is approved by the FDA and as per literature it is widely used as safe drug delivery vehicle to treat several diseases.26 Gelatin is known to possess polyamines on their surface which might be helping to form an amide bond with a carboxylic group containing molecules like rose bengal. RB was found to have a remarkable potentiating effect (extra logs of additional killing up to 6 logs) on aPDT when it is conjugated with gelatin. In this paper, a comparative PDT efficacy of free RB and its



covalent complex with GNP against Gram positive, Gram negative and fungal strains was demonstrated with chloramphenicol as a standard antimicrobial agent. Results and Discussion Synthesis and Characterization of Gelatin Nanoparticles. Gelatin nanoparticles (GNPs) were successfully synthesized using well-established two step desolvation method (SI Figure S1).25 The synthesized nanoparticles were spherical and well dispersed with an average size range of 150-200 nm determined by dynamic light scattering (DLS, SI Figure S2a). This size range of nanoparticles plays an important role to permeate into the infection or tumor site via high permeable capillaries because of enhanced permeability and retention (EPR) effect.27 Positive zeta potential indicating the presence of positively charged groups (NH2) on the surface of GNP is well designed (SI Figure S2c). The polydispersity index (PDI) was found to be 0.11, also implied good monodispersity. The transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis of GNPs revealed the spherical shape of the synthesized nanoparticles (SI Figure 1e & 1f). Quantitatively, 10 mg/mL GNPs were obtained as determined by subtraction method (SI Table S1). Synthesis of RB-GNP. RB-GNP was synthesized via covalent conjugation as well as entrapment or adsorption of RB on BdGNPs surface. The 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was used as cross-coupling agent for this covalent conjugation. RB

molecules possessing carboxylic acid functionality can be covalently attached to the surface of GNP via amide coupling (Figure 1a).28,29 The optimal concentration of GNP to load 100 µg RB was determined. As depicted in the graph (SI Figure S7a), the loading of RB increased above 3 mg/mL GNPs. This might be due to the increased availability of free NH2 groups on the surface of nanoparticles with the increased concentration of GNPs. Less loading (19%) was observed below 3 mg/mL GNPs. It was found that 49% RB was loaded with 3 mg/mL GNPs. Mechanistically, the EDC reacted with the carboxylic group of rose bengal and


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formed an intermediate O-acylisourea active ester, this intermediate coupled with primary amines present in the gelatin nanoparticles and made amide linkage. Initially, EDC and NHS (N-hydroxysuccinimide) were used in the synthesis of RB-GNP as cross-coupling agents. The aggregation of the nanoparticle RB conjugates was noticed when conjugation reaction was performed using both EDC and NHS, however, no aggregation was observed when only EDC was used as coupling agent. Thus, GNPs (10 mg/mL) was used with EDC as a coupling agent. Moreover, gelatin has positive charge at the physiological pH 7.4,30 which is used at the time of conjugation reaction and the attraction between gelatin and negatively charged RB molecules are established. The RB molecules which were not activated by the EDC get adsorbed on the surface or could be internalized into the core of the gelatin. In this instance, both the conjugation as well as adsorption were responsible for the higher loading (95, 96, 95, 90 and 92%) using 90, 182, 274, 364 and 455 µg/mL of RB, respectively (SI Figure S7b). The results were confirmed by comparing with blank reactions in triplicate. A smaller dose of this developed probe is required for the treatment as compared to the conventional doses of the free RB due to its higher drug loading (~95%) in GNPs.4,31 The overall loading of RB on the gelatin nanoparticles was the result of both conjugation as well as physical adsorption. We performed all the synthesis experiments in triplicate in order to determine the reproducibility of the developed nano probes after optimization of various factors. The separated adsorption and conjugation quantification studies have been performed. For this purpose, the separate experiments were carried out without EDC linker to determine the adsorbed amount of RB and with EDC to determine the final loading. The conjugated portion (25%) of RB molecules was calculated by subtracting the entrapped amount of RB (70%) in the absence of EDC from the entrapped amount of RB in the presence of EDC (95%). Determination of Drug Loading of RB-GNP using Absorption Spectroscopy. The GNPs were found to be loaded with 95 ± 1.0% of total RB added. A distinguished loading of 95%



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on the GNPs is praiseworthy and needed a small dose for various biomedical applications. A standard curve of RB was plotted using 2.7 to 15 µg/mL RB (SI Figure S8). Table 1. Determination of percent loading of RB on the GNPs using Absorption spectroscopic method. Rose Bengal (y=0.0778x, R2=0.997)

Parameters

Blank

RB-GNP

Ci (µg/mL)

91

91

Co (µg/mL)

90±0.1

3.4±0.2

Conj./entrap. (%)

1.1±0.3

96±0.2

0

95±1.0

Ld (%)

Ci Initial concentration of RB used in the conjugation reaction. Co concentration of RB in the supernatant after centrifugation, calculated using linear regression equation y = mx+c. Ld percent loading of RB on the surface of the nanoparticle. Conj. Conjugation; entrap. Entrapment. This was calculated using the equation; Conj./entrap. (%) = {[Ci-Co]/[Ci]} x 100; Ld (%) = Conj./entrap. (%)RB-GNP ˗ Conj./entrap. (%)blank Characterization of RB-GNP Size, Charge and Morphology. The size, charge and surface morphology of the nanocarrier is of critical importance for tissue penetration and drug delivery.32 Zeta size and zeta potential analysis are the important tools help to determine the average hydrodynamic particle size and surface charge of nanophototheranostics, respectively. The synthesized RB-GNP exhibited average size 150 to 200 nm. The size of GNPs increased to 176 nm after the conjugation reaction (SI Figure S2b). The low value (0.12) of poly-dispersity index for RB-GNP indicates the mono-dispersive nature of the particles. The decrease in zeta potential from 18 to 7.6 mV


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upon RB incorporation was probably due to amide bond formation between GNPs and RB (reduction in positively charged groups on GNPs surface) (SI Figure S2d). The increased size of GNPs after conjugation of RB was validated by scanning electron microscopy (SEM) (Figure 1b). The surface of GNPs also changed from smooth to wavy as seen in the SEM images of NPT. Transmission electron microscopy (TEM) of nanophototheranostics confirmed that the RB-GNP were spherical in shape with an average size range of 150-200 nm (Figure 1c).

Figure 1. (a) Schematic presentation of the synthesis of RB-GNP. (b) SEM image of RBGNPs (scale 200 nm), inset showing the enlarged image at 50 nm scale. (c) TEM image of RB-GNPs (scale 200 nm) acquired after staining with 2% phosphotungstic acid, inset showing the enlarged image at 50 nm scale.



Physicochemical Characterization. The bare GNPs, RB and RB-GNPs were characterized using attenuated total reflection (ATR) spectroscopy. Clear evidence for the successful reaction of carboxylic (-COOH) functional group in RB with the amine containing GNPs was obtained from ATR overlay. The characteristic peaks of RB at 3006 and 1044 and 952 cm-1 appeared at 3054, 1015, and 951 cm-1 respectively, in the RB-GNP spectra. Infrared stretching at 1633 cm-1 (a characteristic peak of amide-I C=O) was more intense in the RBGNP than bare GNPs, because of additional number of amide bond were formed after the conjugation (SI Figure S3). Circular dichroism (CD) was used to compare the conformational changes in the secondary structure of RB-GNP with gelatin and GNPs in deionized water.33 Gelatin was found in three forms such as crystalline, helical and random coils (unordered). Gelatin dissolved in deionized water at ~37-40 ºC (pH 2-3) shows random coils (unordered).34,35 In the present work, however, gelatin was dissolved in deionized water at ~40 ºC (pH 2.6) and the CD spectra showed a single band below 200 nm which was the characteristic to the random coil conformation (SI Figure S4). Reduction in the intensity of ellipticity due to the formation of covalent bond between RB and gelatin was observed. After conjugation of RB with the GNPs, β-structure increases and minor change in the turns of gelatin conformation was found. The decreased value of random coils confirmed the conjugation (SI Table S2). The patterns of CD analysis were supported by the ATR analysis. The denaturation temperature and the thermo-physical properties of GNPs, RB-GNP and RB were examined by differential scanning calorimetry (DSC, SI Figure S5). For gelatin, at higher temperature (200-230 ºC), the randomly coiled solutions are expected to undergo an additional phase separation yielding an amorphous precipitation, as coherent with the thermodynamics of gelatin solution. The disordered random coil form is stable at higher temperature because the entropy and enthalpy of polymer in this form are higher than the ordered crystalline and/or helical form.36 The endothermic melting temperature (Tm) for


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GNPs was observed at 220 ºC (Cp 10 J/g). The endothermic glass temperature (Tg) for bare RB [86 ºC (Cp 6 J/g) and 100 ºC (Cp 2 J/g)] and endothermic Tm 195 ºC (Cp 2 J/g) were observed. After conjugation with GNPs, the endothermic Tg and Tm were shifted to 145 ºC (Cp 2 J/g) and 163 ºC (Cp 22 J/g), respectively. Due to covalent amide bond formation, more heat was needed as compared to bare RB. The pattern of peaks supports the CD analysis. Lactose was used as cryoprotectant showing two peaks at 147 and 217 ºC and did not show any hindrance in DSC analysis (SI Table S3). The crystalline nature of GNPs, RB-GNP and RB were identified using X-ray diffractometer (XRD, SI Figure S6). A broad hump was observed at 2θ angles of range ~13 to 30º due to amorphous nature of GNPs. RB presents a peak at ~25º suggesting its non-crystalline/semi-crystalline nature. RB-GNP presents a peak at ~25º in addition, the hump of GNPs was shifted to ~16 to 30º. RB-GNP was amorphous, which supported the DSC analysis, resolved by XRD (SI Table S4). The elemental analysis of GNPs was completed by energy dispersive spectroscopy (EDS).37 The bare GNPs showed presence of C, N and O while drug loaded NPs showed changes in the percentage of weight, atom and uncertainty of C, N and O (SI Table S5). These changes were obtained due to the conjugation between GNPs and RB. Release and Dispersion Stability Study. RB-GNP nanoconjugate was further examined for stability and drug release studies.38–40 It was found that the conjugate was stable for at least 7 days under physiological conditions. The RB release profile of the nanoconjugate presents non-significant release (SI Figure S9). The mechanism of slow release is relatively complicated because of covalent bonding. Retardation of the burst release or the slow release is the characteristics of the nano-conjugate and makes this new biodegradable system a good carrier candidate for selective and controlled release of photo sensitizer drug delivery. Moreover, sufficient amount of drug could be delivered at the targeted site through EPR effect because of the nanoparticle size (150-200 nm). Further, after seven days, no significant



change in the size of dispersed RB-GNPs was observed (slight increase in size up to 185 nm). This study suggested that developed nanoconjugates could be stable for a longer time in the systemic environment. Photoluminescence Study of RB-GNP. The fluorescence emission property of developed RB-GNP was demonstrated using fluorescence spectroscopy.24 The aqueous solutions of RB and RB-GNP showed a broad emission band centered on 575 nm (λex 285 nm) at 20 µg/mL. The enhanced fluorescence intensity (4-5 folds) accompanied by a blue shift ~ 12 nm (Figure 2). This was transduced by an increase in the quantum yield from 0.80 (RB) to ~0.96 (RBGNP) (SI Table S6). The absorbance intensity of aqueous/buffered RB-GNP had decreased due to the sedimentation of aggregated species. This was confirmed by the decrease in absorption band in the UV region for a solution containing water as a solvent. Fluorescence enhancement of RB-GNP remained ~4-5 folds more even for the most diluted samples. This suggested that the scattering of the RB-GNP was the major contributor to the fluorescence enhancement. The photo luminescent property of developed probe could be useful in the image guided delivery and their simultaneous determination of the accumulation in various body tissues, if administered systemically. Furthermore, the diagnosis of a particular disease could be possible through the fluorescent imaging using this probe.


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Figure 2. Fluorescence spectra of RB-GNP (red) and RB (blue) (λex = 285 nm). Inset picture is showing the solution of RB-GNP in UV light (365 nm). Determination of Singlet Oxygen Quantum Yield (SOQY) using DPBF Assay. The photophysical property of RB-GNP was determined using green LED irradiation (0.5 W, 500-570 nm).24 The rapid decay in DPBF absorbance (at 410 nm) was observed with free RB, (Figure 3b) whereas comparatively slow decline in DPBF absorbance with RB-GNP was observed (Figure 3a & 3b). SOQY for RB-GNP was found to be 0.39 (SI Table S7). The reduced SOQY in RB-GNP was due to the singlet oxygen quenching in the GNPs. The photophysical properties of RB were intact after conjugation. The advantages of prepared conjugates such as control drug release, biocompatibility and stability at physiological conditions could be beneficial in various applications.

Figure 3. (a) The schematic presentation showing singlet oxygen generation from the developed nanophototheranostic. The corresponding graph showing, the absorbance of DPBF in the solution decreases with irradiation time. (b) An overlay graph showing absorption spectra of DPBF solutions irradiated with green LED at different time intervals. The RB and RB-GNP solutions were mixed separately with DPBF solution before irradiation with light.



The high decay in DPBF absorbance with RB in short time interval as compared to RB-GNP, which means, more amount of RB was loaded on the GNPs. Antimicrobial Photodynamic Therapy. The antimicrobial activity of the RB and RB-GNP were determined using three Gram-negative (Escherichia coli, Serratia marcescens, Pseudomonas putida) one Gram-positive (Bacillus subtilis) and a fungal strain (Candida viswanathii).2,3,41–43 The aqueous RB (5 mM) and RB-GNP (0.089 mM) were separately incubated with each microorganism and the colony counting method (CFUs method) was used to count the viable colonies after incubation, irradiation, and plating (SI Table S8 & S9). The standard antimicrobial drug (chloramphenicol) was used as a reference. The IC50 of RBGNP against E. coli, S. marcescens, P. putida and C. viswanathii was determined using CFU method at 5 to 50 μg/mL (SI Figure S10a-c & S10e), while 0.5 to 20 μg/mL were used to determine IC50 against B. subtilis (SI Figure S10d). The results reveal that the RB-GNP showed efficient antimicrobial activity against various microorganisms with IC50 ranging from 2.6 to 29 nM/mL (Figure 4). The calculated IC50 values of RB-GNP were 22, 21 and 29 μg/mL to inhibit the growth of E. coli, S. marcescens, and P. putida, respectively. The lowest IC50 (2.6 μg/mL) was recorded against B. subtilis. In the case of fungal strain, C. viswanathii IC50 was 21 μg/mL. The inhibitory effect of bare RB and bare GNPs against E. coli, S. marcescens, P. putida, B. subtilis and C. viswanathii was investigated at different concentrations of RB (5 to 50 μg/mL) and GNPs (10 mg/mL). There was no inhibition in microbial growth even at the highest concentration of RB (100 μg/mL) (data not shown). IC50 for chloramphenicol has been calculated after optimization for the respective strains (data not shown).


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Figure 4. CFU counts of various microorganisms (E. coli, S. marcescens, P. putida, B. subtilis and C. vishwanathii) after the treatment with equal concentrations of RB only, Chloramphenicol and RB-GNP. The used concentrations of each test compound (RB only, chloramphenicol and RB-GNP) for various microorganism E. coli, S. marcescens, P. putida, B. subtilis and C. vishwanathii were 22, 21, 29, 2.6 and 21 μg/mL, respectively. The significant growth inhibition was observed after the treatment with RB-GNPs compared to the RB only and chloramphenicol. Further, optimal irradiation time for antimicrobial activity of RB-GNP against different microbial strains were determined using IC50 values for the respective strain (SI Figure S11ae). The optimal irradiation time for 50% inhibition of different microbial strains namely, E. coli, S. marcescens and P. putida, B. subtilis and C. viswanathii were found to be 5, 2, 4, 1 and 2 h, respectively (Table 2). Lowest IC50 with lowest irradiation time was recorded for B. subtilis. It might be due to the positive charge over the cell membrane as RB-GNP is easily attached to it. Hence, it gets excited easily and leads to cell death. Here, we developed a costeffective formulation which could be applicable to treat surface infections (acne, burn, lesions



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etc.) using a cheap light source such as LEDs. A low powered LED light (0.5 Watt) was used which required more time for the excitation of RB-GNP and sufficient production of reactive oxygen species. Using the higher intensity of a particular wavelength the same results could be obtained in very short time (5-10 min). Moreover, the light irradiated by a laser could be able to penetrate deeper into the body tissue which could be beneficial to excite this molecule present in the areas of internal infections. Table 2. Determination of IC50 of RB-GNP Cell

Irradiation

Chloramphenicol

inhibition

time (h) at

IC50 (μg/mL)

using RB at

RB-GNP

(n=3)

RB-GNP IC50

IC50 value

value (n=3)

(n=3)

RB-GNP Microorganism

IC50

(MTCC No.)

(μg/mL) (n=3)

Escherichia coli (443)

22 ± 2

73 ± 1.8

No inhibition

5 ± 0.1

Serratia marcescens (4301)

22 ± 1.7

75 ± 1.4

No inhibition

2 ± 0.3

Pseudomonas putida (1237)

29 ± 2

81 ± 2

No inhibition

4 ± 0.8

Bacillus subtilis (1427)

2.6 ± 1.2

65 ± 1.3

No inhibition

1.5 ± 0.1

Candida viswanathii (5158)

21 ± 2.1

75 ± 1.7

No inhibition

2 ± 0.2

Furthermore, to confirm the antimicrobial effect of the developed Bd-NPTs, the confocal laser scanning microscopy (CLSM) of treated and untreated bacterial samples was used. Propidium iodide (PI), a well-known dead cell specific stain, can only enter into the membrane compromised dead microbial cells rather than the viable cells. The intact cell membrane it produces red fluorescence (λem 645 nm) upon excitation wavelength 488 nm.29,38,41,44,45 To determine the cell viability properly washed treated and untreated microbial


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cells were stained with PI and simultaneously observed under the CLSM. The CLSM images showed most of the treated bacteria emitted red fluorescence (λem 645 nm) when excited using 488 nm laser, whereas, the untreated and treated with RB bacteria did not emit red fluorescence (Figure 5). Moreover, the CLSM analysis suggested that the developed BdNPTs interacted with the microbial cell membrane and were responsible for the membrane denaturation by the generation of singlet oxygen species upon light irradiation.

42

This study

confers the mechanism of antimicrobial photodynamic therapy using Bd-NPTs. All these results suggested that RB-GNP could be an effective and alternative treatment for the various multi-antibiotic-resistant infections. Moreover, the higher loading and controlled release properties of these developed probes make them beneficial to deliver the photosensitizer at the tumor site in various cancers. Because of this behavior, RB-GNPs could be used to kill cancerous cells in the solid tumours. Mechanistic Aspects of aPDT. To confirm the mechanism of antimicrobial photodynamic therapy, DNA cleavage study has been performed. The cleavage of super coiled pBR 322 DNA was studied using agarose gel electrophoresis.41 Gel images indicate that the DNA was not cleaved by RB and RB-GNP even after 5 h of incubation and irradiation (SI Figure S12) suggesting that the RB and RB-GNP were likely to alter the structure of cell membrane. This might be most plausible reason for its biological activity. The SEM analyses further supported this observation showing damage in the cell membrane of Gram-negative (Escherichia coli), Gram-positive (Bacillus subtilis) and yeast (Candida viswanathii) cells when they are incubated with RB-GNP (Figure 6). It has been reported that the reactive oxygen species causes the damage of cellular membrane through the oxidation or peroxidation of lipid or other biomolecules present in the cell membrane and simultaneously responsible for the cell demise.46 However, we proved the interaction of RB-GNPs with the outer surface of the microbial cells via scanning electron microscopy (SEM).



Figure 5. CLSM images of E. coli (a) S. marcescens (b) P. putida (c) B. subtilis (d) and C. viswanathii (e), before and after PDT treatment (scale 10 μm). Panel ‘i’ represents the images


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of untreated cells and showing the presence of mostly unstained viable cells. Panels ‘ii’and ‘iii’, represents the images of E. Coli, S. marcescens, P. putida, B. subtilis and C. viswanathii treated with RB and RB-GNP, respectively. The microbial cells corresponding to the treated groups showing the significant red fluorescence at λem 645 nm. The significant fluorescence in the merged panel suggested the remarkable cell death after the PDT treatment as compared to untreated cells.

Figure 6. The characterization of the interaction of RB-GNPs with the microorganisms and simultaneous cellular membrane damage after aPDT using SEM analysis, here, (a, d and g) SEM image of E. coli (scale 5 µm), B. subtilis (scale 10 µm) and C. vishwanathii (scale 10



µm) without any treatment. Inset SEM image (scale 100 nm, 5 µm, and 5µm, respectively) showing intact cell membrane. Similarly, (b, e and h) E. coli (scale 5 µm), B. subtilis (scale 10 µm) and C. vishwanathii (scale 10 µm) incubated with RB and inset showing intact cell membrane (scale 100 nm, 5 µm and 5µm, respectively); (c, f and i) E. coli (scale 5 µm), B. subtilis (scale 10 µm) and C. vishwanathii (scale 10 µm) incubated with RB-GNP and inset showing damage in cell membrane (scale 100 nm, 5 µm and 5µm, respectively). Experimental Section Synthesis, Optimization and Characterization of RB-GNP. The RB-GNP was synthesized using chemical

cross

linking by

carbodiimide

[1-Ethyl-3-(3-dimethylaminopropyl)

carbodiimide; EDC] coupling based reaction.47 In brief, solutions of rose bengal (20 µL from 5 mg/mL stock solution) and EDC (2.4 µL from 5 mg/mL stock solution) were mixed in a micro centrifuge tube. After 20 minutes, gelatin nanoparticles (10 mg) were added and stirred at 25 °C for 18 h in dark condition (150 rpm). The conjugated product was then centrifuged using an ultracentrifuge (Eppendorf) at 16900 × g and 25 °C for 1 h. The UV-absorption spectrum (556 nm) of the resulted supernatant was recorded to determine the presence of rose bengal using a double beam UV-visible spectrophotometer (Lab India UV-3200). The concentration of rose bengal in the supernatant was calculated using the linear equation. Simultaneously, the percent loading of RB into the gelatin nanoparticles was calculated using the following equation: Ld (%) = [(Total RB added–RB in the supernatant)/Total RB added] x 100…… (i) The standard calibration curve and linear regression equation for the quantitative analysis of rose bengal were determined by recording the OD of its different concentrations (2.5 to 25 μg/mL) at 556 nm (SI Figure S6). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 10 mM) was added in the flasks containing different concentrations of RB (90 to 900 μg),


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separately. After 20 minutes of reaction, GNPs (10 mg) were added and the reaction volume was made up to 1 mL using deionized water. The reaction mixture was then incubated at 25 ºC (150 rpm) in dark for 18 h. To optimize the GNPs concentration, the flasks containing different amount of GNPs (100 µL to 1 mL) from 10 mg/mL GNPs stock were added to separate flasks containing the optimized concentration of activated RB (100 µg) with EDC (10 mM) in each. The reaction mixtures were incubated at 25 ºC (150 rpm) for 18 h in dark. The synthesized RB-GNPs were then characterized using various techniques. To estimate the particle size and zeta potential, a diluted colloidal solution of RB-GNP (50 µL, diluted up to 1 mL) was prepared in deionized water and subjected to dynamic light scattering (DLS) analysis using Zetasizer (Malvern, UK) with a fixed wavelength of 532 nm at 25 ºC with 90º detection angle. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed to determine the surface morphology of RB-GNP. The conjugation on the surface of the gelatin nanoparticles was confirmed by attenuated total reflection (ATR) spectroscopy (Perkin Elmer, USA). The lyophilized RB-GNP was subjected to ATR spectroscopic analysis along with the rose bengal and bare gelatin nanoparticles. Circular dichroism measurements were carried out to determine the changes in secondary structure of gelatin after conjugation on a JASCO 715 spectropolarimeter (JASCO 715; Japan) using a quartz cell of 0.1 cm path length. The spectra were recorded at 25 ºC and were the average of a series of three scans made at 10 nm intervals in the 190-250 nm. The concentration of sample (RB-GNP, gelatin, and GNPs) was 100 µg/mL in deionized water. Ellipticity is reported as the mean residue ellipticity [θ]R [deg cm2 dmol-1]. The endothermic curves of RBGNP were recorded from 25-300 ºC using a differential scanning calorimetry (DSC, Mettler Toledo) at heating rate 10 ºC /min in nitrogen atmosphere after cooling with liquid nitrogen. The x-ray powder diffraction pattern of lyophilized RB-GNP was obtained with an X-ray



diffractometer (Bruker D8) to determine the crystallinity of RB-GNP. The XRD patterns were recorded at 2θ. Release and Dispersion Stability Study. Following a general procedure, the in vitro release study of RB-loaded gelatin nanoparticles was performed at 37 ºC in dark using dynamic dialysis technique.40 Briefly, the synthesized RB-GNP preparation (10 mg/mL, donor solution) with conjugation efficiency 95% was kept in a dialysis membrane with a molecular weight cut-off of 10 kDa and dialyzed against 20 mL phosphate buffer saline (PBS, pH 7.4, receiver solution). Receiver solution (1 mL) was withdrawn at a regular time interval (0 h, 3 h, 6 h, 12 h up to 7 days) and the same volume of PBS was added. The absorbance of the withdrawn receiver solution was measured at 556 nm and concentrations of rose bengal were calculated using the standard regression equation. The percent drug release was calculated using the following equation: R (%) = Volume of sample withdrawn (mL)/bath volume (v) × P (t – 1) + Pt………(ii) where, R (%) is cumulative percentage release, Pt is the percentage release at time t, and P (t – 1) is the percentage release previous to‘t’ Further, to determine the stability of synthesized RB-GNPs, the dispersion of RB-GNPs was collected from the dialysis bag after seven days and subjected to Zeta size analysis. Photoluminescence. The fluorescence spectrum of a diluted colloidal solution of RB-GNPs was recorded using Varian carry eclipse (Agilent Technologies, USA) fluorescence spectrophotometer. The RB-GNP solution was excited at 285 nm (excitation slit 5 nm) and the emission spectrum was recorded at 400-800 nm (emission slit 5 nm). The fluorescence quantum yield of RB-GNPs was determined using the following equation: 𝑄𝑄𝑢𝑢 = 𝑄𝑄𝑠𝑠 ×

𝐼𝐼𝑠𝑠

𝐼𝐼𝑢𝑢

×

2 𝐴𝐴𝑠𝑠 𝜂𝜂𝑢𝑢

𝐴𝐴𝑢𝑢 𝜂𝜂𝑠𝑠2

……….(iii)


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where, u and s represent unknown and standard, respectively. I = integrated fluorescence intensity, A = absorbance at λex, η = refractive index of solvent. Singlet Oxygen Quantification Assay. Singlet oxygen generation was assayed using a known method with some modifications.24 In brief, 5 mg/mL RB stock solution was prepared in deionized water. 1,3-diphenylisobenzofuran (DPBF, 3 mg/mL) solution was prepared in DMSO and kept in dark. In a cuvette containing DPBF solution (5 µL), the solution of RB (5 µL) was added and the final volume was made upto 1 mL using DMSO in dark. The cuvette containing reaction mixture was then irradiated with green light (500-570 nm, 0.5 W). Simultaneously, the absorbance of DPBF at 410 nm was recorded at different time of intervals (0, 0.5, 1, 5, 10 and 20 min). For test, 25 µL solution of RB-GNP (1 mg/mL) was added and the absorbance of DPBF (at 410 nm) was monitored at 0, 5, 10, 15, 30, 45, 60, 75 and 90 min. Singlet oxygen quantum yields were calculated using following equation: 𝜑𝜑(𝑈𝑈) = 𝜑𝜑(𝑆𝑆𝑆𝑆)

𝑆𝑆(𝑈𝑈)

𝑆𝑆(𝑆𝑆𝑆𝑆)

……….(iv)

where, U and St denotes unknown and standard, respectively. S represents slope. Antimicrobial Photodynamic Therapy. A stock solution of RB-GNP (0.089 mM) based on the RB content was prepared in sterilized ultrapure water. The strains (Escherichia coli 443, Serratia marcescens 4301, Pseudomonas putida 1237, Bacillus subtilis 1427 and Candida viswanathii 5158) were procured from the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh (India). The bacterial cells of Escherichia coli, Serratia marcescens, Pseudomonas putida and Bacillus subtilis were grown in Luria Bertani (LB) broth while fungal strain Candida viswanathii was grown in yeast peptone dextrose (YPD) medium in aerobic conditions at 37 ºC (200 rpm) until 0.6 optical density at 600 nm. The culture broth with a concentration of 1-3×108 CFU/mL was pelleted by centrifugation (10 min, 7000 × g) at room temperature, the supernatant was discarded, and



the cells were resuspended in 1 mL sterilized ultrapure water. In order to determine the optimized inhibitory concentrations of test compounds, microbial strains E. coli, S. marcescens, P. putida and C. viswanathii were incubated with the different concentrations (5, 10, 20, 30, 40 and 50 µg/mL) of the RB and RB-GNPs while B. subtilis was incubated with a lower concentration (0.5, 2.5, 5, 10, 15 and 20 µg/mL) of RB and RB-GNP. The treated and non-treated cells were incubated for 5 h in dark at 150 rpm. After incubation, sterile microcentrifuge tubes containing cells were illuminated with Green LED (500-570 nm, 0.5 W) for 5 h and the IC50 value of RB-GNPs for each microorganism was determined. The light illumination time was further optimized for various test microorganisms using optimized IC50 value of RB-GNP by irradiating the samples for 1-5 h in separate experiments. In all these experiments, the lamp was placed at a distance of 5 cm above the sample. After illumination, 100 µL cell suspension was serially diluted 106 times and plated on the divided Petri plates (LB Broth-Agar plates for bacteria E. coli, S. marcescens, P. putida and B. subtilis and YPD agar plates for fungal yeast C. viswanathii). The plates were incubated in aerobic condition at 37 °C in dark for overnight. The colonies were counted in each plate and the survival of cells was determined from the ratio of CFU/mL of the illuminated solution and the light and dark control. All the experiments were performed in triplicate (SI Table S8 and Table S9). Confocal Laser Scanning Microscopy (CLSM). Bacterial cell viability of treated and untreated samples was determined using propidium iodide (PI) dead cell staining dye.24 After the PDT treatment, the bacterial cells were washed and resuspended in phosphate buffer saline (pH 7.4), separately. From each sample (diluted 105x), 100 µL cell suspension was dispensed into a 24 well plate, separately. A solution of propidium iodide (50 µL, 15 µM) was added to each well and incubated for 15 min at 22 °C in dark with mild shaking. The bacterial samples were analyzed under CLSM (Olympus, Microscope FV 1000 SPD, Japan)


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using a 10x lens with PI filter, where laser excitation and emission wavelengths were adjusted to 488 and 645 nm, respectively. The respective images for each sample were acquired using FluoView® FV10-ASW Viewer software (ver.4.2b). DNA Cleavage Study. The cleavage of super coiled pBR 322 DNA was studied by agarose gel electrophoresis.41 The pBR 322 DNA (0.2 μg/mL) was treated with two different concentrations (5 and 10 μg) of RB and RB-GNP in four separate micro centrifuge tubes. Aqueous solutions of circular plasmid DNA (pBR 322) were added in four separate microcentrifuge tubes containing RB (5 and 10 μg) and RB-GNP (5 and 10 μg) in Tris buffer (pH = 7.2) and incubated at 25 °C. Two sets of the reaction mixture in duplicate were incubated for 1 and 5 h followed by irradiation using a green LED (500-570 nm, 0.5 W) at 25 °C for 1 and 5 h, respectively. The samples were then analyzed by agarose gel electrophoresis (75 V, 1 h) in Tris-Borate-EDTA (TBE) buffer (pH 8.3, 1x) to determine the DNA cleavage ability of the RB and RB-GNP. Determination of Microbial Membrane Damage. The microbial cell membrane damage was determined using scanning electron microscopy (SEM). Treated and untreated microbial cell suspensions were diluted to 106x and simultaneously vortexed to break the agglomeration. A small droplet was then deposited on the flat carbon tape using a micropipette. The samples were subjected to SEM analysis after proper drying. Conclusions A biocompatible RB-GNP nanoconjugate was developed with 95% photosensitizer loading. Photo-physical properties of the nanoconjugates were determined using a conventional light source (LED, 500-570 nm) showing the higher value of singlet oxygen quantum yield (39%). Approximate ~4-5 fold enhanced fluorescence intensity of RB-GNP (FQY 0.96) compared to bare rose bengal indicates respectable photoluminescence properties of nanoconjugates. The



antibacterial photodynamic activity of RB-GNP suggested significant killing of Grampositive bacteria (IC50 2.6 μg/mL). It also showed antimicrobial effect on Gram-negative bacteria and yeast with IC50 values ranging from 21 to 29 μg/mL. The mechanistic studies suggest that the RB-GNPs are likely to interact with the outer cell membrane by altering the membrane structure through oxidation or peroxidation of lipids and other biomolecules present in the cellular membrane. From the various applications point of view of RB-GNPs, it may be concluded that the probe could be used for various biomedical applications such as drug delivery, fluorescence imaging, cancer eradication, etc. Supporting Information Supporting information is available containing: Materials and Experimental, surface functionalization of gelatin nanoparticles, optimization of drug loading reaction; characterization of Bd-NPTs by ATR spectroscopy; CD; XRD; DSC; PL studies and singlet oxygen generation studies; Biocompatibility study; Antimicrobial assay. Conflicts of Interest We have no conflict of interest to declare. Acknowledgements SK acknowledges the funding support by the Department of Biotechnology (DBT), Govt. of India to carry out this work. NST acknowledges the financial support from DST INSPIRE fellowship (Govt. of India). JB gratefully acknowledges financial support from the Department of Science and Technology, Govt. of India, through DST Women Scientists Scheme A (WOS-A) Scheme. References (1)

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TOC Development of Gelatin Nanoparticle Based Biodegradable Phototheranostic Agents: Advanced System to Treat Infectious Diseases Seema Kirar 1, Neeraj S. Thakur 2, Joydev K. Laha 3, Jayeeta Bhaumik 1,# and Uttam C. Banerjee1, 2,*


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Development of Gelatin Nanoparticle-Based Biodegradable

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