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J Photochem Photobiol B. Author manuscript; available in PMC 2017 July 27. Published in final edited form as: J Photochem Photobiol B. 2016 February ; 155: 60–65. doi:10.1016/j.jphotobiol.2015.12.007.
Photophysical characterization of anticancer drug Valrubicin in rHDL nanoparticles and its use as an imaging agent Sunil Shah1, Rahul Chib1, Sangram Raut2, Jaclyn Bermudez3, Nirupama Sabnis1, Divya Duggal1, Joseph.D. Kimball2, Andras G Lacko1, Zygmunt Gryczynski1,2, and Ignacy Gryczynski1,* 1Department
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of Cell Biology and Immunology, Center for Fluorescence Technologies and Nanomedicine, UNT Health Science Center, Fort Worth, TX, USA 2Department
of Physics and Astronomy, Texas Christian University, Fort Worth, TX, USA
3Cell
Biology and Immunology, North Texas Eye Research Institute, UNT Health Science Center, Fort Worth, TX, USA 4Department
of Integrative Physiology and Anatomy, UNT Health Science Center, Fort Worth, TX,
USA
Abstract
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Nanoparticles are target-specific drug delivery agents that are increasingly used in cancer therapy to enhance bioavailability and to reduce off target toxicity of anti-cancer agents. Valrubicin is an anti-cancer drug, currently approved only for vesicular bladder cancer treatment because of its poor water solubility. On the other hand, valrubicin carrying reconstituted high density lipoprotein (rHDL) nanoparticles appear ideally suited for extended applications, including systemic cancer chemotherapy. We determined selected fluorescence properties of the free (unencapsulated) drug vs. valrubicin incorporated into rHDL nanoparticles. We have found that upon encapsulation into rHDL nanoparticles the quantum yield of valrubicin fluorescence increased six fold while its fluorescence lifetime increased about 2 fold. Accordingly, these and potassium iodide (KI) quenching data suggest that upon incorporation, valrubicin is localized deep in the interior of the nanoparticle, inside the lipid matrix. Fluorescence anisotropy of the rHDL valrubicin nanoparticles was also found to be high along with extended rotational correlation time. The fluorescence of valrubicin could also be utilized to assess its distribution upon delivery to prostate cancer (PC3) cells. Overall the fluorescence properties of the rHDL: valrubicin complex reveal valuable novel characteristics of this drug delivery vehicle that may be particularly applicable when used in systemic (intravenous) therapy.
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Keywords Valrubicin; rHDL nanoparticles; fluorescence spectroscopy; cancer; confocal imaging *
Corresponding Authors: [email protected], [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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1. Introduction
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Valrubicin or AD-32 (C34H36F3NO13) is an anti-cancer drug which is an N-trifluoroacetyl, 14-valerate derivative of the anthracycline doxorubicin1. Cells exposed to valrubicin in vitro show that the drug and its metabolites hinder breaking and resealing of DNA strands by topoisomerase II, and also inhibit incorporation of nucleosides into DNA and RNA2,3. This may lead to chromosomal damage, and also result in arresting the cell cycle between the S and G2 phase, showing that valrubicin has antineoplasticity1,2. Compared with doxorubicin (4–10mg/Kg), the optimal dose of valrubicin (40–120mg/Kg) achieved survival times that were 1.1–14 fold higher in mice with experimentally induced leukemia, lymphoma or lung carcinoma1,4. However, like chemotherapeutic agents, valrubicin has toxic side effects and unrestricted tissue distribution. In addition, because of its poor water solubility and biocompatibility, its use is restricted to carcinoma in situ of bladder5. Thus, managing toxicity and solubility of chemotherapeutic agents is important in tackling cancerous tumors6. To overcome this problem, drug delivery via nanotechnology is one of the innovative methods in the field of cancer therapeutics5. Targeted drug delivery has the advantage of reducing drug interaction with healthy tissue thereby reducing off target toxicity. Carriers such as lipoprotein type nanoparticles are especially suited for lipophilic drug delivery, and provide an alternate option to using traditional carriers like emulsions, liposomes and other polymers7,8.
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Reconstituted high-density lipoprotein (rHDL) nano-carriers minimize off target toxicity compared to traditional carriers for drug delivery9,10. High-density lipoproteins (HDL) are a major class of plasma proteins with a shielded hydrophobic core that is very suitable to accommodate hydrophobic drugs, including valrubicin5,11. It is known from earlier findings that tumor cells over express scavenger receptor type B1 (SR-B1), to “scavenge” rHDL particles presumably to maintain a high growth rate11–13. In addition to SR-B1 mediated rHDL homing, of the rHDL drug delivery system has the potential to improve the transport of anti-cancer agents by avoiding the membrane associated pump system responsible for multi-drug resistance6,14.
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Having known its usefulness in drug delivery area, it is important to characterize the optical properties of Valrubicin that is incorporated into rHDL in comparison to its free (nonencapsulated) form. In this current study, we evaluated the optical properties of free and rHDL valrubicin to address problems in the context of therapeutic effect of the drug: delivery across a range of biological barriers to the intracellular site of action, and transport without altering the physiochemical properties of the drug15,16. We also studied the uptake of free and rHDL valrubicin using confocal microscopy to evaluate its potential as a theranostic agent10, given its specificity towards cancer cells over expressing SR-B1 receptors11,12.
2. Materials Sodium cholate, egg yolk, phosphatidyl choline (PC), free cholesterol, cholesterol oleate, potassium bromide (KBr), isopropyl thiogalactoside (IPTG), dimethyl sulfoxide (DMSO), J Photochem Photobiol B. Author manuscript; available in PMC 2017 July 27.
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Trioton X-100, and thrombin cleavage kit were purchased from Sigma-Aldrich Corporation, St. Louis, Mo. NZYCM was obtained from Teknova, Hollister, CA. Bacterial protein extraction reagent and bicinchoninic acid (BCA) protein assay kits were purchased from Thermo Scientific, Rockford, IL. AD-32 was provided by Dr Mervyn Israel, University of Tennessee Health Science Center, Memphis, TN. Low protein binding durapore membrane syringe filters were purchased from Millipore Ltd, Billerica, MA. Potassium iodide was purchased from Sigma-Aldrich Corporation, St. Louis, Mo.
3. Methods 3.1 Preparation of AD-32 containing nanoparticles: Isolation and purification of recombinant apoA-1
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These were performed essentially as described by Ryan et. al.17 Briefly, 500ml NZYCM media with 50μg/ml ampicillin at 37°C was used to culture BL21 (De3) pLysS cells bearing the pNFXex plasmid. When the optical density of the culture reached 0.6 at 600nm, addition of IPTG to a final concentration of 0.5mM was used to induce apoA-I synthesis. After three hours, the bacteria were pelleted by centrifugation and disrupted by bacterial protein extraction reagent. The cell lysate was centrifuged at 20,000 g for 30 minutes at 4°C. For purification, the supernatant fraction was mixed with an equal volume of phosphate-buffered saline (PBS) containing 6M guanidine hydrochloric acid (HCL), and applied to a 5ml bed volume His-Trap affinity column. The thrombin cleavage kit from Sigma-Aldrich was used to remove the N-terminal His-Tag extension. The isolated apo A-I was then dialyzed against tris (hydroxymethyl) aminomethane (Tris)-buffered saline containing 1 mM benzamidine for 16 hours at 4° C. The dialyzed sample was filter-sterilized (0.2 µm) and stored at 4° C until use5.
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3.2 Preparation of rHDL/AD-32 complexes with apo A-1
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This was accomplished by a procedure developed earlier in Dr. Lacko’s laboratory. Briefly, a mixture of egg yolk PC in CHCl3 with free cholesterol (FC) and cholesteryl oleate (CE) was prepared with a molar ratio of ApoA1: FC: CE: PC = 1:5:1.3:1.15 M. The lipid mixture of PC, CE, and FC along with the drug (Ad-32) were dried under nitrogen to a thin film and dispersed in 60μL DMSO. Apo A-I (5 mg) and 140 μL sodium cholate (from a stock of 100mM) were added to the above mixture and the volume made up to 2 mL with Trisethylenediaminetetraacetic acid (EDTA) buffer. The lipid/protein/cholate mixture was then incubated for 12 hours at 4°C, followed by dialysis against 2L of PBS for 48 hours, including three buffer changes in the first 12 hours. The preparations were then centrifuged at 1000 rpm for 2 minutes and then sterilized using 0.2μm syringe filter. The preparation was kept in the dark at 4°C until used5. 3.3 Spectroscopic measurements UV-Vis absorption and fluorescence spectra were obtained using a Cary 50 bio UV–Visible spectrophotometer (Varian Inc.) and Cary Eclipse spectrofluorometer (Varian Inc.) respectively. All the measurements were done in 1cm X 1cm cuvette at room temperature unless otherwise mentioned. 10% DMSO was added to the total volume of free valrubicin in
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PBS to improve solubility of the drug. The quantum yield for free Valrubicin and rHDL Valrubicin was calculated by using rhodamine B in ethanol as a reference. To measure the emission spectra, free and rHDL valrubicin were excited at 495 nm, and their emission observed from 530 nm to 800 nm using a 515 nm long-pass filter. For excitation spectra, free and rHDL valrubicin’s emission was observed at 620nm and excitation was scanned from 330 nm to 580 nm using a 590 nm long-pass filter on the emission side.
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In order to determine the location and accessibility of valrubicin within rHDL nanoparticles in comparison to free valrubicin, we measured the quenching of valrubicin using potassium iodide which is a known quencher. The concentration range of the quencher used was from 0M – 800mM. Emission spectra were scanned from 530 nm-800 nm with 495 nm excitation and a 515 nm long pass filter. The points were fit using a linear fit, and the Stern-Volmer quenching constant was calculated using the following formula: (1)
Where F0/F is the ratio of the initial intensity to the intensity at varying quencher concentrations, K is the Stern-Volmer quenching constant and [Q] defines the quencher concentration.
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To further determine the stability of valrubicin encapsulated in rHDL nanoparticles and to understand more about the microenvironment of both free and rHDL valrubicin, temperature dependent steady-state anisotropy was measured using Varian Cary Peltier single cell holder, model DBS-SPV-1, on the Cary Eclipse spectrofluorometer (Varian Inc.) The temperature range for this instrument is from −10C to 110 C with an accuracy of +/− 0.1C. It achieves heating and cooling by electrical heat pumps with a circulated water bath. The scan conditions for the spectra collected were the same as the conditions mentioned above for collecting the emission spectra of valrubicin, and were done using a manual polarizer. The G factor was measured and incorporated in the anisotropy calculation using the following formula:
(2)
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Time resolved anisotropy and fluorescence lifetime was measured on a FluoTime 200 fluorometer (PicoQuant, Inc.) using 470 nm laser diode. The fluorometer is equipped with an ultrafast micro channel plate (MCP) PMT from Hamamatsu, Inc. and emission was observed at 600 nm with vertical and horizontal polarizer positions on emission side. Anisotropy decays were analyzed with a multi-exponential fitting model in FluoFit3 program from PicoQuant, Inc (Germany) using following equation:
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(3)
The fluorescence lifetimes were measured in magic angle condition and data analyzed using FluoFit3 program from PicoQuant, Inc (Germany) using multi-exponential fitting model:
(4)
The quality of the fits in lifetime and anisotropy decays’ analysis were judged by the chi square (χR2) values and by the quality of the residuals and autocorrelation of these residuals.
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3.4 Con-focal Imaging
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Uptake of rHDL-valrubicin nanoparticles into cells was visualized using the intrinsic fluorescent property of valrubicin under confocal microscope. To observe the internalization of nanoparticles under a confocal microscope, prostate cancer cell line PC-3 was grown in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cellular uptake of free valrubicin and rHDL-valrubicin was determined using a confocal microscope (Zeiss LSM 510 META attached to a Zeiss Axiovert 200 inverted microscope) (Carl Zeiss Micro Imaging, Inc., Thornwood, NY). For this experiment, cells were placed on sterile cover slips in a 6-well tissue culture plate and incubated at 37°C and 5% CO2 until they reached sub-confluent levels. The cells were then exposed to 10 μg/ml concentration of valrubicin in free and rHDL formulation. After the desired incubation time, the treated cells were fixed with standard paraformaldehyde (4%) and fixed using a gold antifade mounting agent with 4’-6-diamidino-2-phenylindole (DAPI). The slides were viewed under the microscope to determine the extent of intracellular nanoparticle uptake. Images were analyzed using Zeiss LSM image browser software.
4. Results and Discussions 4.1 Absorption spectra The absorption spectra of free and rHDL valrubicin are presented in Figure 1. There is no observable change in the absorption pattern of free valrubicin in 10% DMSO vs. the rHDL encapsulated valrubicin. We did observe the presence of a scattering component as seen on the blue edge of the spectrum perhaps due to the particulate nature of the system.
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4.2 Steady state fluorescence measurements 4.2.1 Excitation and emission spectra—Normalized excitation and emission spectra of free and rHDL valrubicin are presented in figure 2. Free valrubicin showed Stokes shift of 100 nm, compared to 89nm exhibited by rHDL valrubicin. Valrubicin appears to be in a stable environment leading to decreased energy losses and hence the smaller Stokes shift. The emission spectra for free and rHDL valrubicin were similar. Excitation spectra show rHDL valrubicin to have a more “structured” spectrum compared to free valrubicin,
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suggesting reduced vibrational losses. The quantum yield of rHDL valrubicin was 9.11% and for free valrubicin was 1.53% indicating an almost 6 fold increase in quantum yields upon encapsulating valrubicin in rHDL, perhaps due to the shielding of the molecule by the lipid environment resulting decreased non-radiative losses.
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4.2.2 Stern-Volmer quenching plot—To further check the accessibility of the encapsulated dye in rHDL particles, quenching studies using potassium iodide (KI) were carried out. The Stern-Volmer plot for the quenching experiment performed is shown in figure 3. KI was used as a quencher, with the concentration range from 0 mM to 800 mM. The samples were prepared in PBS at room temperature with increasing quencher concentrations, and their emission spectra collected using the conditions described in the methods section. The change in emission intensity due to KI quenching was linear as fitted using line equation, suggesting collisional quenching by potassium iodide. The linear plot thus further confirms only one type of quenching. The Stern-Volmer quenching constants for free valrubicin and rHDL valrubicin respectively were calculated to be 58.6 × 10−7 M−1 and 6.71 × 10−7 M−1. Because the Stern-Volmer quenching constant defines the sensitivity of a fluorophore to a quencher, free valrubicin being unshielded is quenched more rapidly and at lower quencher concentrations compared to valrubicin incorporated into and thus shielded in the rHDL nanoparticles. These findings also support the concept that valrubicin (a lipophilic molecule) is deeply buried inside the hydrophobic core matrix of the rHDL particles and perhaps only a very small population which is towards the outer surface or physically adsorbed onto surface monolayer of the rHDL particles is accessible to the KI.
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4.2.3 Steady state temperature dependent anisotropy—Being encapsulated in large molecular weight (~180,000) lipoprotein nanoparticles, one would expect high fluorescence anisotropy for rHDL valrubicin. Steady-state anisotropy measurements performed with rHDL and free valrubicin over a range of temperatures are shown in figure 4. Because of the high (50–70%) lipid content of the rHDL nanoparticles, we anticipated that the fluorescence parameters (including anisotropy) would undergo a phase change at higher temperatures. In order to test this hypothesis, we measured rHDL valrubicin anisotropy from 10°C to 100°C. Free valrubicin’s anisotropy reached almost zero around 70°C, without any trace of phase change unlike other phospholipid particles such as DMPC. This could be due to the presence of other biomolecules in the rHDL structure such as Apo-A1 protein and cholates which may alter the physical properties of these particles. rHDL has higher anisotropy overall compared to free valrubicin. This can be explained by the larger size of rHDL nanoparticles compared to free drug. The change in anisotropy can be used in-vitro to study the drug release without the need of any dialysis membrane as there is large difference in anisotropy of free and encapsulated valrubicin. The anisotropy will start at higher number at initial time and will drop as a function of time. 4.3. Time resolved measurements 4.3.1 Fluorescence lifetime measurements—The fluorescence decay measurements of free and rHDL valrubicin are shown in figure 5. Intensity decays in both cases were multi-exponential and heterogeneous in nature. Average amplitude weighted lifetime of free valrubicin is 0.39 ns and 0.75 ns for the rHDL/valrubicin complex. Lifetime of valrubicin
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almost doubled in HDL environment. Average intensity weighted lifetime for rHDL valrubicin particle is 1.20 ns compared to the value for free valrubicin, which has a lifetime of 0.89ns (Table 1). The calculated radiative rates (kr) for free and encapsulated valrubicin are 0.038 × 10−9 s-1 and 0.12 × 10−9 s-1. However, the non-radiative rates were 2.5 × 10−9 s-1 and 1.2 × 10−9 s-1 for free and encapsulated valrubicin respectively. The radiative rate changed more than 3 times while the non-radiative rate halved in case of rHDL nanoparticles apparently owing to stabilization (restricted motion) of the valrubicin molecules in the rHDL matrix.
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4.3.2 Time resolved anisotropy measurements—Free valrubicin exhibited a rapid anisotropy decay/loss and faster correlation time. Comparison of the anisotropy decays of free valrubicin and rHDL valrubicin is shown in figure 6. A slower motion and slower decay was observed in case of rHDL valrubicin particles. Correlation time in case of free valrubicin particle was 0.64 ns with R0 0.17. More than half the total anisotropy was lost, apparently due to the very fast motion that is outside the instrumental resolution. On the other hand, in case of the rHDL valrubicin particles, the observed correlation time was 522.95 ns (large uncertainty) for the first and major component and 0.69 ns for the second component, with R0 of 0.24. The large correlation time in case of rHDL particles may be ascribed to the global motion of the particles while the fast component is likely to be the result of the local motion of the dye/drug. 4.4 Confocal microscopy
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Studying these fluorescence properties will help study the drug delivery in order to understand the interaction of rHDL with cells and tissues. Moreover, one can look at the cellular fate of these particles using microscopy. Here we show that valrubicin itself can be used as a fluorescent marker. The free valrubicin has low quantum yield and is not regarded as a good fluorophore for this purpose. However, encapsulated valrubicin shows about 10% quantum yield and it becomes interesting to use it in fluorescence spectroscopy and microscopy studies. Figure 7 shows the confocal images of PC3 cells with free and encapsulated valrubicin. Top 3 horizontal panels shows the control cells and only fluorescence observed was from DAPI stained nucleus. Further, free valrubicin also shows considerable fluorescence as evidenced by the diffuse green fluorescence throughout the cytoplasm. Moreover, in case of rHDL particles one can see the diffuse fluorescence as well as the bright punctuate spots which could be internalized rHDL particles. One can also observe the particles close to the cell wall that are attached either on the outer surface or internalized. Here we did not follow the kinetics of rHDL internalization or drug release; however, one can follow these processes by using the fluorescence signal from valrubicin.
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5. Conclusions In summary, we have provided the fluorescence based studies onto Valrubicin as a fluorophore. It has low quantum yield in aqueous environment and it goes up several times when encapsulated in lipid particles. Moreover, similar changes were observed in terms of fluorescence lifetime. Fluorescence anisotropy was found to be high in case of particles. Accessibility of the dye was studied using the KI quenching and found that it’s been buried
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deeply inside the lipid matrix core. Fluorescence of Valrubicin was used to observe the distribution of particles and free dye inside a cancer cell line using con-focal microscopy. Moreover, one can use the fluorescence properties of this dye/drug to its advantage while developing it as anti-cancer drug therapy.
Acknowledgments This work was supported by the NIH grant R01EB12003 (Z.G), NSF grant CBET-1264608 (I.G), and Cancer Prevention and Research Institute of Texas grant DP150091 (AGL).
References
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1. Onrust SV, Lamb HM. Valrubicin. Drugs Aging. 1999; 15(1):69–75. [PubMed: 10459733] 2. Blum, R., Garnick, M., Israel, M., Panellos, G., Henderson, I., Frei, E, III. New drugs in cancer chemotherapy. Springer; 1981. Preclinical rationale and phase I clinical trial of the adriamycin analog, AD 32; p. 7-15. 3. Chow KC, Macdonald TL, Ross WE. DNA binding by epipodophyllotoxins and N-acyl anthracyclines: Implications for mechanism of topoisomerase II inhibition. Mol Pharmacol. 1988; 34(4):467–473. [PubMed: 2845248] 4. Israel M, Modest EJ, Frei E 3rd. N-trifluoroacetyladriamycin-14-valerate, an analog with greater experimental antitumor activity and less toxicity than adriamycin. Cancer Res. 1975; 35(5):1365– 1368. [PubMed: 1054622] 5. Sabnis N, Nair M, Israel M, McConathy WJ, Lacko AG. Enhanced solubility and functionality of valrubicin (AD-32) against cancer cells upon encapsulation into biocompatible nanoparticles. Int J Nanomedicine. 2012; 7:975–983. [doi]. DOI: 10.2147/IJN.S28029 [PubMed: 22393294] 6. Lacko AG, Nair M, Paranjape S, Johnson S, McConathy WJ. A novel delivery system for targeted cancer therapy. Technology in Cancer Research and Treatment. 2005; 21:179–183. 7. Freitas C, Müller RH. Correlation between long-term stability of solid lipid nanoparticles (SLN™) and crystallinity of the lipid phase. European Journal of Pharmaceutics and Biopharmaceutics. 1999; 47(2):125–132. http://dx.doi.org/10.1016/S0939-6411(98)00074-5. [PubMed: 10234536] 8. Uner M, Yener G. Importance of solid lipid nanoparticles (SLN) in various administration routes and future perspectives. Int J Nanomedicine. 2007; 2(3):289–300. [PubMed: 18019829] 9. Lacko AG, Nair M, Paranjape S, Mooberry L, McConathy WJ. Trojan horse meets magic bullet to spawn a novel, highly effective drug delivery model. Chemotherapy. 2006; 52(4):171–173. doi: 93268 [pii]. [PubMed: 16691026] 10. Sabnis N, Lacko AG. Drug delivery via lipoprotein-based carriers: Answering the challenges in systemic therapeutics. Therapeutic delivery. 2012; 3(5):599–608. [PubMed: 22834404] 11. Shahzad MM, Mangala LS, Han HD, et al. Targeted delivery of small interfering RNA using reconstituted high-density lipoprotein nanoparticles. Neoplasia. 2011; 13(4):309–IN8. [PubMed: 21472135] 12. McConathy WJ, Paranjape S, Mooberry L, Buttreddy S, Nair M, Lacko AG. Validation of the reconstituted high-density lipoprotein (rHDL) drug delivery platform using dilauryl fluorescein (DLF). Drug delivery and translational research. 2011; 1(2):113–120. [PubMed: 25788110] 13. Lacko AG, Nair M, Prokai L, McConathy WJ. Prospects and challenges of the development of lipoprotein-based formulations for anti-cancer drugs. 2007 14. Lehne G. P-glycoprotein as a drug target in the treatment of multidrug resistant cancer. Curr Drug Targets. 2000; 1(1):85–99. [PubMed: 11475537] 15. Banerjee C, Maiti S, Mustafi M, et al. Effect of encapsulation of curcumin in polymeric nanoparticles: How efficient to control ESIPT process? Langmuir. 2014; 30(36):10834–10844. [PubMed: 25148375] 16. Kamaly N, Xiao Z, Valencia PM, Radovic-Moreno AF, Farokhzad OC. Targeted polymeric therapeutic nanoparticles: Design, development and clinical translation. Chem Soc Rev. 2012; 41(7):2971–3010. [PubMed: 22388185]
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17. Ryan RO, Forte TM, Oda MN. Optimized bacterial expression of human apolipoprotein AI. Protein Expr Purif. 2003; 27(1):98–103. [PubMed: 12509990]
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Highlights •
Anti-cancer drug valrubicin has the potential to be a good theranostic agent
•
rHDL nanoparticles effectively shield hydrophobic valrubicin.
•
Use of rHDL nanoparticles can reduce toxicity and improve diagnostic time.
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rHDl and free valrubicin have intrinsic fluorescence that can be used for imaging.
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Compared to valrubicin, rHDL nanoparticles have longer lifetime and quantum yield.
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Figure 1.
Absorption spectra of rHDL valrubicin and free valrubicin.
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Figure 2.
Normalized excitation and emission spectra of free valrubicin (left panel) and rHDL valrubicin (right panel).
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Figure 3.
Stern-Volmer plot for free and rHDL valrubicin.
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Figure 4.
Temperature dependent steady state anisotropy for rHDL and free valrubicin.
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Figure 5.
Fluorescence intensity decay for free and rHDL valrubicin using a 470 nm laser diode for excitation.
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Figure 6.
Time resolved anisotropy for free and rHDl valrubicin.
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Figure 7.
Confocal microscopy of free and rHDL valrubicin using cell lines.
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rHDL valrubicin
Free valrubicin
Sample
1.15
0.27 2.87
1.10 0.41
0.07 0.11
----
τ4
0.76
0.12 0.09
0.77
α2
α1
τ3
τ1
τ2
Amplitudes
Lifetime (ns)
0.09
0.10
α3
0.04
----
α4
1.20
0.89
τint.
0.75
0.39
τamp.
Average lifetime (ns)
Analysis of free and rHDL valrubicin fluorescence intensity decay with multi-exponential model.
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Table 1 Shah et al. Page 18
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Author Manuscript 0.17 0.24
rHDL valrubicin
0.15
0.17 0.09
-522.95
0.64 ±0.024 0.69±0.034
--
Φ2
Φ1
R2
R0
R1
Correlation time (ns)
Anisotropy
Free Valrubicin
Sample
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Time resolved anisotropy data for free and rHDL valrubicin.
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Table 2 Shah et al. Page 19
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J Photochem Photobiol B. Author manuscript; available in PMC 2017 July 27. Published in final edited form as: J Photochem Photobiol B. 2016 February ; 155: 60–65. doi:10.1016/j.jphotobiol.2015.12.007.
Photophysical characterization of anticancer drug Valrubicin in rHDL nanoparticles and its use as an imaging agent Sunil Shah1, Rahul Chib1, Sangram Raut2, Jaclyn Bermudez3, Nirupama Sabnis1, Divya Duggal1, Joseph.D. Kimball2, Andras G Lacko1, Zygmunt Gryczynski1,2, and Ignacy Gryczynski1,* 1Department
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of Cell Biology and Immunology, Center for Fluorescence Technologies and Nanomedicine, UNT Health Science Center, Fort Worth, TX, USA 2Department
of Physics and Astronomy, Texas Christian University, Fort Worth, TX, USA
3Cell
Biology and Immunology, North Texas Eye Research Institute, UNT Health Science Center, Fort Worth, TX, USA 4Department
of Integrative Physiology and Anatomy, UNT Health Science Center, Fort Worth, TX,
USA
Abstract
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Nanoparticles are target-specific drug delivery agents that are increasingly used in cancer therapy to enhance bioavailability and to reduce off target toxicity of anti-cancer agents. Valrubicin is an anti-cancer drug, currently approved only for vesicular bladder cancer treatment because of its poor water solubility. On the other hand, valrubicin carrying reconstituted high density lipoprotein (rHDL) nanoparticles appear ideally suited for extended applications, including systemic cancer chemotherapy. We determined selected fluorescence properties of the free (unencapsulated) drug vs. valrubicin incorporated into rHDL nanoparticles. We have found that upon encapsulation into rHDL nanoparticles the quantum yield of valrubicin fluorescence increased six fold while its fluorescence lifetime increased about 2 fold. Accordingly, these and potassium iodide (KI) quenching data suggest that upon incorporation, valrubicin is localized deep in the interior of the nanoparticle, inside the lipid matrix. Fluorescence anisotropy of the rHDL valrubicin nanoparticles was also found to be high along with extended rotational correlation time. The fluorescence of valrubicin could also be utilized to assess its distribution upon delivery to prostate cancer (PC3) cells. Overall the fluorescence properties of the rHDL: valrubicin complex reveal valuable novel characteristics of this drug delivery vehicle that may be particularly applicable when used in systemic (intravenous) therapy.
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Keywords Valrubicin; rHDL nanoparticles; fluorescence spectroscopy; cancer; confocal imaging *
Corresponding Authors: [email protected], [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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1. Introduction
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Valrubicin or AD-32 (C34H36F3NO13) is an anti-cancer drug which is an N-trifluoroacetyl, 14-valerate derivative of the anthracycline doxorubicin1. Cells exposed to valrubicin in vitro show that the drug and its metabolites hinder breaking and resealing of DNA strands by topoisomerase II, and also inhibit incorporation of nucleosides into DNA and RNA2,3. This may lead to chromosomal damage, and also result in arresting the cell cycle between the S and G2 phase, showing that valrubicin has antineoplasticity1,2. Compared with doxorubicin (4–10mg/Kg), the optimal dose of valrubicin (40–120mg/Kg) achieved survival times that were 1.1–14 fold higher in mice with experimentally induced leukemia, lymphoma or lung carcinoma1,4. However, like chemotherapeutic agents, valrubicin has toxic side effects and unrestricted tissue distribution. In addition, because of its poor water solubility and biocompatibility, its use is restricted to carcinoma in situ of bladder5. Thus, managing toxicity and solubility of chemotherapeutic agents is important in tackling cancerous tumors6. To overcome this problem, drug delivery via nanotechnology is one of the innovative methods in the field of cancer therapeutics5. Targeted drug delivery has the advantage of reducing drug interaction with healthy tissue thereby reducing off target toxicity. Carriers such as lipoprotein type nanoparticles are especially suited for lipophilic drug delivery, and provide an alternate option to using traditional carriers like emulsions, liposomes and other polymers7,8.
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Reconstituted high-density lipoprotein (rHDL) nano-carriers minimize off target toxicity compared to traditional carriers for drug delivery9,10. High-density lipoproteins (HDL) are a major class of plasma proteins with a shielded hydrophobic core that is very suitable to accommodate hydrophobic drugs, including valrubicin5,11. It is known from earlier findings that tumor cells over express scavenger receptor type B1 (SR-B1), to “scavenge” rHDL particles presumably to maintain a high growth rate11–13. In addition to SR-B1 mediated rHDL homing, of the rHDL drug delivery system has the potential to improve the transport of anti-cancer agents by avoiding the membrane associated pump system responsible for multi-drug resistance6,14.
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Having known its usefulness in drug delivery area, it is important to characterize the optical properties of Valrubicin that is incorporated into rHDL in comparison to its free (nonencapsulated) form. In this current study, we evaluated the optical properties of free and rHDL valrubicin to address problems in the context of therapeutic effect of the drug: delivery across a range of biological barriers to the intracellular site of action, and transport without altering the physiochemical properties of the drug15,16. We also studied the uptake of free and rHDL valrubicin using confocal microscopy to evaluate its potential as a theranostic agent10, given its specificity towards cancer cells over expressing SR-B1 receptors11,12.
2. Materials Sodium cholate, egg yolk, phosphatidyl choline (PC), free cholesterol, cholesterol oleate, potassium bromide (KBr), isopropyl thiogalactoside (IPTG), dimethyl sulfoxide (DMSO), J Photochem Photobiol B. Author manuscript; available in PMC 2017 July 27.
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Trioton X-100, and thrombin cleavage kit were purchased from Sigma-Aldrich Corporation, St. Louis, Mo. NZYCM was obtained from Teknova, Hollister, CA. Bacterial protein extraction reagent and bicinchoninic acid (BCA) protein assay kits were purchased from Thermo Scientific, Rockford, IL. AD-32 was provided by Dr Mervyn Israel, University of Tennessee Health Science Center, Memphis, TN. Low protein binding durapore membrane syringe filters were purchased from Millipore Ltd, Billerica, MA. Potassium iodide was purchased from Sigma-Aldrich Corporation, St. Louis, Mo.
3. Methods 3.1 Preparation of AD-32 containing nanoparticles: Isolation and purification of recombinant apoA-1
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These were performed essentially as described by Ryan et. al.17 Briefly, 500ml NZYCM media with 50μg/ml ampicillin at 37°C was used to culture BL21 (De3) pLysS cells bearing the pNFXex plasmid. When the optical density of the culture reached 0.6 at 600nm, addition of IPTG to a final concentration of 0.5mM was used to induce apoA-I synthesis. After three hours, the bacteria were pelleted by centrifugation and disrupted by bacterial protein extraction reagent. The cell lysate was centrifuged at 20,000 g for 30 minutes at 4°C. For purification, the supernatant fraction was mixed with an equal volume of phosphate-buffered saline (PBS) containing 6M guanidine hydrochloric acid (HCL), and applied to a 5ml bed volume His-Trap affinity column. The thrombin cleavage kit from Sigma-Aldrich was used to remove the N-terminal His-Tag extension. The isolated apo A-I was then dialyzed against tris (hydroxymethyl) aminomethane (Tris)-buffered saline containing 1 mM benzamidine for 16 hours at 4° C. The dialyzed sample was filter-sterilized (0.2 µm) and stored at 4° C until use5.
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3.2 Preparation of rHDL/AD-32 complexes with apo A-1
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This was accomplished by a procedure developed earlier in Dr. Lacko’s laboratory. Briefly, a mixture of egg yolk PC in CHCl3 with free cholesterol (FC) and cholesteryl oleate (CE) was prepared with a molar ratio of ApoA1: FC: CE: PC = 1:5:1.3:1.15 M. The lipid mixture of PC, CE, and FC along with the drug (Ad-32) were dried under nitrogen to a thin film and dispersed in 60μL DMSO. Apo A-I (5 mg) and 140 μL sodium cholate (from a stock of 100mM) were added to the above mixture and the volume made up to 2 mL with Trisethylenediaminetetraacetic acid (EDTA) buffer. The lipid/protein/cholate mixture was then incubated for 12 hours at 4°C, followed by dialysis against 2L of PBS for 48 hours, including three buffer changes in the first 12 hours. The preparations were then centrifuged at 1000 rpm for 2 minutes and then sterilized using 0.2μm syringe filter. The preparation was kept in the dark at 4°C until used5. 3.3 Spectroscopic measurements UV-Vis absorption and fluorescence spectra were obtained using a Cary 50 bio UV–Visible spectrophotometer (Varian Inc.) and Cary Eclipse spectrofluorometer (Varian Inc.) respectively. All the measurements were done in 1cm X 1cm cuvette at room temperature unless otherwise mentioned. 10% DMSO was added to the total volume of free valrubicin in
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PBS to improve solubility of the drug. The quantum yield for free Valrubicin and rHDL Valrubicin was calculated by using rhodamine B in ethanol as a reference. To measure the emission spectra, free and rHDL valrubicin were excited at 495 nm, and their emission observed from 530 nm to 800 nm using a 515 nm long-pass filter. For excitation spectra, free and rHDL valrubicin’s emission was observed at 620nm and excitation was scanned from 330 nm to 580 nm using a 590 nm long-pass filter on the emission side.
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In order to determine the location and accessibility of valrubicin within rHDL nanoparticles in comparison to free valrubicin, we measured the quenching of valrubicin using potassium iodide which is a known quencher. The concentration range of the quencher used was from 0M – 800mM. Emission spectra were scanned from 530 nm-800 nm with 495 nm excitation and a 515 nm long pass filter. The points were fit using a linear fit, and the Stern-Volmer quenching constant was calculated using the following formula: (1)
Where F0/F is the ratio of the initial intensity to the intensity at varying quencher concentrations, K is the Stern-Volmer quenching constant and [Q] defines the quencher concentration.
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To further determine the stability of valrubicin encapsulated in rHDL nanoparticles and to understand more about the microenvironment of both free and rHDL valrubicin, temperature dependent steady-state anisotropy was measured using Varian Cary Peltier single cell holder, model DBS-SPV-1, on the Cary Eclipse spectrofluorometer (Varian Inc.) The temperature range for this instrument is from −10C to 110 C with an accuracy of +/− 0.1C. It achieves heating and cooling by electrical heat pumps with a circulated water bath. The scan conditions for the spectra collected were the same as the conditions mentioned above for collecting the emission spectra of valrubicin, and were done using a manual polarizer. The G factor was measured and incorporated in the anisotropy calculation using the following formula:
(2)
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Time resolved anisotropy and fluorescence lifetime was measured on a FluoTime 200 fluorometer (PicoQuant, Inc.) using 470 nm laser diode. The fluorometer is equipped with an ultrafast micro channel plate (MCP) PMT from Hamamatsu, Inc. and emission was observed at 600 nm with vertical and horizontal polarizer positions on emission side. Anisotropy decays were analyzed with a multi-exponential fitting model in FluoFit3 program from PicoQuant, Inc (Germany) using following equation:
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(3)
The fluorescence lifetimes were measured in magic angle condition and data analyzed using FluoFit3 program from PicoQuant, Inc (Germany) using multi-exponential fitting model:
(4)
The quality of the fits in lifetime and anisotropy decays’ analysis were judged by the chi square (χR2) values and by the quality of the residuals and autocorrelation of these residuals.
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3.4 Con-focal Imaging
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Uptake of rHDL-valrubicin nanoparticles into cells was visualized using the intrinsic fluorescent property of valrubicin under confocal microscope. To observe the internalization of nanoparticles under a confocal microscope, prostate cancer cell line PC-3 was grown in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cellular uptake of free valrubicin and rHDL-valrubicin was determined using a confocal microscope (Zeiss LSM 510 META attached to a Zeiss Axiovert 200 inverted microscope) (Carl Zeiss Micro Imaging, Inc., Thornwood, NY). For this experiment, cells were placed on sterile cover slips in a 6-well tissue culture plate and incubated at 37°C and 5% CO2 until they reached sub-confluent levels. The cells were then exposed to 10 μg/ml concentration of valrubicin in free and rHDL formulation. After the desired incubation time, the treated cells were fixed with standard paraformaldehyde (4%) and fixed using a gold antifade mounting agent with 4’-6-diamidino-2-phenylindole (DAPI). The slides were viewed under the microscope to determine the extent of intracellular nanoparticle uptake. Images were analyzed using Zeiss LSM image browser software.
4. Results and Discussions 4.1 Absorption spectra The absorption spectra of free and rHDL valrubicin are presented in Figure 1. There is no observable change in the absorption pattern of free valrubicin in 10% DMSO vs. the rHDL encapsulated valrubicin. We did observe the presence of a scattering component as seen on the blue edge of the spectrum perhaps due to the particulate nature of the system.
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4.2 Steady state fluorescence measurements 4.2.1 Excitation and emission spectra—Normalized excitation and emission spectra of free and rHDL valrubicin are presented in figure 2. Free valrubicin showed Stokes shift of 100 nm, compared to 89nm exhibited by rHDL valrubicin. Valrubicin appears to be in a stable environment leading to decreased energy losses and hence the smaller Stokes shift. The emission spectra for free and rHDL valrubicin were similar. Excitation spectra show rHDL valrubicin to have a more “structured” spectrum compared to free valrubicin,
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suggesting reduced vibrational losses. The quantum yield of rHDL valrubicin was 9.11% and for free valrubicin was 1.53% indicating an almost 6 fold increase in quantum yields upon encapsulating valrubicin in rHDL, perhaps due to the shielding of the molecule by the lipid environment resulting decreased non-radiative losses.
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4.2.2 Stern-Volmer quenching plot—To further check the accessibility of the encapsulated dye in rHDL particles, quenching studies using potassium iodide (KI) were carried out. The Stern-Volmer plot for the quenching experiment performed is shown in figure 3. KI was used as a quencher, with the concentration range from 0 mM to 800 mM. The samples were prepared in PBS at room temperature with increasing quencher concentrations, and their emission spectra collected using the conditions described in the methods section. The change in emission intensity due to KI quenching was linear as fitted using line equation, suggesting collisional quenching by potassium iodide. The linear plot thus further confirms only one type of quenching. The Stern-Volmer quenching constants for free valrubicin and rHDL valrubicin respectively were calculated to be 58.6 × 10−7 M−1 and 6.71 × 10−7 M−1. Because the Stern-Volmer quenching constant defines the sensitivity of a fluorophore to a quencher, free valrubicin being unshielded is quenched more rapidly and at lower quencher concentrations compared to valrubicin incorporated into and thus shielded in the rHDL nanoparticles. These findings also support the concept that valrubicin (a lipophilic molecule) is deeply buried inside the hydrophobic core matrix of the rHDL particles and perhaps only a very small population which is towards the outer surface or physically adsorbed onto surface monolayer of the rHDL particles is accessible to the KI.
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4.2.3 Steady state temperature dependent anisotropy—Being encapsulated in large molecular weight (~180,000) lipoprotein nanoparticles, one would expect high fluorescence anisotropy for rHDL valrubicin. Steady-state anisotropy measurements performed with rHDL and free valrubicin over a range of temperatures are shown in figure 4. Because of the high (50–70%) lipid content of the rHDL nanoparticles, we anticipated that the fluorescence parameters (including anisotropy) would undergo a phase change at higher temperatures. In order to test this hypothesis, we measured rHDL valrubicin anisotropy from 10°C to 100°C. Free valrubicin’s anisotropy reached almost zero around 70°C, without any trace of phase change unlike other phospholipid particles such as DMPC. This could be due to the presence of other biomolecules in the rHDL structure such as Apo-A1 protein and cholates which may alter the physical properties of these particles. rHDL has higher anisotropy overall compared to free valrubicin. This can be explained by the larger size of rHDL nanoparticles compared to free drug. The change in anisotropy can be used in-vitro to study the drug release without the need of any dialysis membrane as there is large difference in anisotropy of free and encapsulated valrubicin. The anisotropy will start at higher number at initial time and will drop as a function of time. 4.3. Time resolved measurements 4.3.1 Fluorescence lifetime measurements—The fluorescence decay measurements of free and rHDL valrubicin are shown in figure 5. Intensity decays in both cases were multi-exponential and heterogeneous in nature. Average amplitude weighted lifetime of free valrubicin is 0.39 ns and 0.75 ns for the rHDL/valrubicin complex. Lifetime of valrubicin
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almost doubled in HDL environment. Average intensity weighted lifetime for rHDL valrubicin particle is 1.20 ns compared to the value for free valrubicin, which has a lifetime of 0.89ns (Table 1). The calculated radiative rates (kr) for free and encapsulated valrubicin are 0.038 × 10−9 s-1 and 0.12 × 10−9 s-1. However, the non-radiative rates were 2.5 × 10−9 s-1 and 1.2 × 10−9 s-1 for free and encapsulated valrubicin respectively. The radiative rate changed more than 3 times while the non-radiative rate halved in case of rHDL nanoparticles apparently owing to stabilization (restricted motion) of the valrubicin molecules in the rHDL matrix.
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4.3.2 Time resolved anisotropy measurements—Free valrubicin exhibited a rapid anisotropy decay/loss and faster correlation time. Comparison of the anisotropy decays of free valrubicin and rHDL valrubicin is shown in figure 6. A slower motion and slower decay was observed in case of rHDL valrubicin particles. Correlation time in case of free valrubicin particle was 0.64 ns with R0 0.17. More than half the total anisotropy was lost, apparently due to the very fast motion that is outside the instrumental resolution. On the other hand, in case of the rHDL valrubicin particles, the observed correlation time was 522.95 ns (large uncertainty) for the first and major component and 0.69 ns for the second component, with R0 of 0.24. The large correlation time in case of rHDL particles may be ascribed to the global motion of the particles while the fast component is likely to be the result of the local motion of the dye/drug. 4.4 Confocal microscopy
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Studying these fluorescence properties will help study the drug delivery in order to understand the interaction of rHDL with cells and tissues. Moreover, one can look at the cellular fate of these particles using microscopy. Here we show that valrubicin itself can be used as a fluorescent marker. The free valrubicin has low quantum yield and is not regarded as a good fluorophore for this purpose. However, encapsulated valrubicin shows about 10% quantum yield and it becomes interesting to use it in fluorescence spectroscopy and microscopy studies. Figure 7 shows the confocal images of PC3 cells with free and encapsulated valrubicin. Top 3 horizontal panels shows the control cells and only fluorescence observed was from DAPI stained nucleus. Further, free valrubicin also shows considerable fluorescence as evidenced by the diffuse green fluorescence throughout the cytoplasm. Moreover, in case of rHDL particles one can see the diffuse fluorescence as well as the bright punctuate spots which could be internalized rHDL particles. One can also observe the particles close to the cell wall that are attached either on the outer surface or internalized. Here we did not follow the kinetics of rHDL internalization or drug release; however, one can follow these processes by using the fluorescence signal from valrubicin.
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5. Conclusions In summary, we have provided the fluorescence based studies onto Valrubicin as a fluorophore. It has low quantum yield in aqueous environment and it goes up several times when encapsulated in lipid particles. Moreover, similar changes were observed in terms of fluorescence lifetime. Fluorescence anisotropy was found to be high in case of particles. Accessibility of the dye was studied using the KI quenching and found that it’s been buried
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deeply inside the lipid matrix core. Fluorescence of Valrubicin was used to observe the distribution of particles and free dye inside a cancer cell line using con-focal microscopy. Moreover, one can use the fluorescence properties of this dye/drug to its advantage while developing it as anti-cancer drug therapy.
Acknowledgments This work was supported by the NIH grant R01EB12003 (Z.G), NSF grant CBET-1264608 (I.G), and Cancer Prevention and Research Institute of Texas grant DP150091 (AGL).
References
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1. Onrust SV, Lamb HM. Valrubicin. Drugs Aging. 1999; 15(1):69–75. [PubMed: 10459733] 2. Blum, R., Garnick, M., Israel, M., Panellos, G., Henderson, I., Frei, E, III. New drugs in cancer chemotherapy. Springer; 1981. Preclinical rationale and phase I clinical trial of the adriamycin analog, AD 32; p. 7-15. 3. Chow KC, Macdonald TL, Ross WE. DNA binding by epipodophyllotoxins and N-acyl anthracyclines: Implications for mechanism of topoisomerase II inhibition. Mol Pharmacol. 1988; 34(4):467–473. [PubMed: 2845248] 4. Israel M, Modest EJ, Frei E 3rd. N-trifluoroacetyladriamycin-14-valerate, an analog with greater experimental antitumor activity and less toxicity than adriamycin. Cancer Res. 1975; 35(5):1365– 1368. [PubMed: 1054622] 5. Sabnis N, Nair M, Israel M, McConathy WJ, Lacko AG. Enhanced solubility and functionality of valrubicin (AD-32) against cancer cells upon encapsulation into biocompatible nanoparticles. Int J Nanomedicine. 2012; 7:975–983. [doi]. DOI: 10.2147/IJN.S28029 [PubMed: 22393294] 6. Lacko AG, Nair M, Paranjape S, Johnson S, McConathy WJ. A novel delivery system for targeted cancer therapy. Technology in Cancer Research and Treatment. 2005; 21:179–183. 7. Freitas C, Müller RH. Correlation between long-term stability of solid lipid nanoparticles (SLN™) and crystallinity of the lipid phase. European Journal of Pharmaceutics and Biopharmaceutics. 1999; 47(2):125–132. http://dx.doi.org/10.1016/S0939-6411(98)00074-5. [PubMed: 10234536] 8. Uner M, Yener G. Importance of solid lipid nanoparticles (SLN) in various administration routes and future perspectives. Int J Nanomedicine. 2007; 2(3):289–300. [PubMed: 18019829] 9. Lacko AG, Nair M, Paranjape S, Mooberry L, McConathy WJ. Trojan horse meets magic bullet to spawn a novel, highly effective drug delivery model. Chemotherapy. 2006; 52(4):171–173. doi: 93268 [pii]. [PubMed: 16691026] 10. Sabnis N, Lacko AG. Drug delivery via lipoprotein-based carriers: Answering the challenges in systemic therapeutics. Therapeutic delivery. 2012; 3(5):599–608. [PubMed: 22834404] 11. Shahzad MM, Mangala LS, Han HD, et al. Targeted delivery of small interfering RNA using reconstituted high-density lipoprotein nanoparticles. Neoplasia. 2011; 13(4):309–IN8. [PubMed: 21472135] 12. McConathy WJ, Paranjape S, Mooberry L, Buttreddy S, Nair M, Lacko AG. Validation of the reconstituted high-density lipoprotein (rHDL) drug delivery platform using dilauryl fluorescein (DLF). Drug delivery and translational research. 2011; 1(2):113–120. [PubMed: 25788110] 13. Lacko AG, Nair M, Prokai L, McConathy WJ. Prospects and challenges of the development of lipoprotein-based formulations for anti-cancer drugs. 2007 14. Lehne G. P-glycoprotein as a drug target in the treatment of multidrug resistant cancer. Curr Drug Targets. 2000; 1(1):85–99. [PubMed: 11475537] 15. Banerjee C, Maiti S, Mustafi M, et al. Effect of encapsulation of curcumin in polymeric nanoparticles: How efficient to control ESIPT process? Langmuir. 2014; 30(36):10834–10844. [PubMed: 25148375] 16. Kamaly N, Xiao Z, Valencia PM, Radovic-Moreno AF, Farokhzad OC. Targeted polymeric therapeutic nanoparticles: Design, development and clinical translation. Chem Soc Rev. 2012; 41(7):2971–3010. [PubMed: 22388185]
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Highlights •
Anti-cancer drug valrubicin has the potential to be a good theranostic agent
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rHDL nanoparticles effectively shield hydrophobic valrubicin.
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Use of rHDL nanoparticles can reduce toxicity and improve diagnostic time.
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rHDl and free valrubicin have intrinsic fluorescence that can be used for imaging.
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Compared to valrubicin, rHDL nanoparticles have longer lifetime and quantum yield.
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Figure 1.
Absorption spectra of rHDL valrubicin and free valrubicin.
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Figure 2.
Normalized excitation and emission spectra of free valrubicin (left panel) and rHDL valrubicin (right panel).
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Figure 3.
Stern-Volmer plot for free and rHDL valrubicin.
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Figure 4.
Temperature dependent steady state anisotropy for rHDL and free valrubicin.
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Figure 5.
Fluorescence intensity decay for free and rHDL valrubicin using a 470 nm laser diode for excitation.
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Figure 6.
Time resolved anisotropy for free and rHDl valrubicin.
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Figure 7.
Confocal microscopy of free and rHDL valrubicin using cell lines.
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rHDL valrubicin
Free valrubicin
Sample
1.15
0.27 2.87
1.10 0.41
0.07 0.11
----
τ4
0.76
0.12 0.09
0.77
α2
α1
τ3
τ1
τ2
Amplitudes
Lifetime (ns)
0.09
0.10
α3
0.04
----
α4
1.20
0.89
τint.
0.75
0.39
τamp.
Average lifetime (ns)
Analysis of free and rHDL valrubicin fluorescence intensity decay with multi-exponential model.
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Table 1 Shah et al. Page 18
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Author Manuscript 0.17 0.24
rHDL valrubicin
0.15
0.17 0.09
-522.95
0.64 ±0.024 0.69±0.034
--
Φ2
Φ1
R2
R0
R1
Correlation time (ns)
Anisotropy
Free Valrubicin
Sample
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Time resolved anisotropy data for free and rHDL valrubicin.
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Table 2 Shah et al. Page 19
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