Unveiling the Mode of Interaction of Berberine ... - ACS Publications

Subscriber access provided by ORTA DOGU TEKNIK UNIVERSITESI KUTUPHANESI

Article

Unveiling the Mode of Interaction of Berberine Alkaloid in Different Supramolecular Confined Environments: Interplay of Surface Charge Between Nano-Confined Charged Layer and DNA Niloy Kundu, Arpita Roy, Debasis Banik, and Nilmoni Sarkar J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 12 Jan 2016 Downloaded from http://pubs.acs.org on January 12, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

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

The Journal of Physical Chemistry

Unveiling the Mode of Interaction of Berberine Alkaloid in different Supramolecular Confined Environments: Interplay of Surface Charge between Nano-Confined Charged Layer and DNA Niloy Kundu, Arpita Roy, Debasis Banik, Nilmoni Sarkar* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, WB, India E-mail: [email protected] Fax: 91-3222-255303 Abstract In this manuscript, we demonstrate a detail characterization of binding interaction of Berberine Chloride (BBCl) with Calf-Thymus DNA (CT-DNA) in buffer solution as well as in two differently charged reverse micelles (RMs). The photophyscial properties of this alkaloid have been modulated within these microheterogeneous bio-assemblies. The mode of binding of this alkaloid with DNA is of debate till date. However, fluorescence spectroscopic measurements, Circular Dichroism (CD) measurement and temperature dependent study unambiguously establish that BBCl partially intercalate into the DNA base pairs. The nonplanarity imposed by partial saturation in their structure causes the non-classical types of intercalation into DNA. Besides the intercalation, electrostatic interactions also play a significant role in the binding between BBCl and DNA. DNA structure turn into condense form after encapsulation into RMs which is followed by the CD spectra and microscopy study. The probe location and dynamics in the nanopool of the RMs is depended on the electrostatic interaction between the charged surfactants and cationic Berberine. The structural alteration of CT-DNA from B form to condense form and the interplay of surface charge between RMs and DNA determine the interaction between the alkaloid and DNA in RMs. Time resolved study and fluorescence anisotropy measurements successfully provide the binding interaction of BBCl in the nanopool of the RMs in absence and in presence of DNA. This study motivates us to judge further the potential applicability of this alkaloid in other biological systems or other biomimicking organized assemblies.

1 ACS Paragon Plus Environment


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

1. Introduction: Berberine Chloride, a well known quaternary isoquinoline alkaloid is a naturally occurring medicine. It is mainly used as a drug for the treatment of diarrhea and gastroenteritis dates to several thousand years ago1 and the anti-malarial, anti-hypertensive, anti-tumor and antiinflammatory activity of this alkaloid is well reported.2-8 Recent studies also indicate that they are the promising candidate for the treatment of cancer9 and Alzheimer’s diseases10 and it also helps to reduce the amount of cholesterol and lipid in both plasma and liver.11 Beside these, the alkaloid is also effective in eliminating bacterial plasmids.12 Based on these information and other evidences, it was postulated that Berberine Chloride can also be utilized to the binding to the extra chromosomal DNA and it can shows biological activities by blocking transcription or replication.13 Protoberberines are also well known DNA binder.14-15 The interaction between the small molecules and DNA is important for the development of the effective therapeutic agents1617

and in recent years, it became the subject of active scientific research in different fields such as

medicinal chemistry, clinical medicine, life science etc.18-20 It is well known that small molecules interact with the DNA via three different possible modes: ionic, groove and intercalation binding. The factors which determine the interaction are quite complex in terms of their reaction modes and kinetic mechanism. Therefore, to design and synthesize new efficient DNA targeted antitumor or anticancer agent it is important to consider these factors. The mode of binding between protoberebrine and DNA is of debate till date. The small molecules which are mostly aromatic, planar and polycyclic insert between the base pairs of DNA and this phenomenon is known as intercalation. Coralyne Chloride which constitute the unsaturated napthoisoquinoline chromophore similar to Berberine Chloride, intercalate between DNA base pairs.21 Therefore, it was first suggested that Berberine Chloride also bind to the DNA by intercalation mode following the classical intercalation model.22 However, in contrast to planar Coralyne Chloride, Berberine Chloride possesses partial saturation in their structure and therefore, ring 4 is slightly twisted out of the ring 1-2 plane (scheme-1). Fluorescence study first suggested that the binding mechanism is groove binding rather than the intercalation binding.23 However, partial intercalation is also suggested from the 1H NMR study.24 Beside these experimental debates, some theoretical models also recommend the partial intercalation of Berberine Chloride into the DNA.14 Recently, Li et al proposed the intercalation binding as well as the electrostatic interaction between the Berberine Chloride and Herring sperm DNA via spectroscopic 2 ACS Paragon Plus Environment

Page 2 of 37

Page 3 of 37

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


methods.25 Due to such discrepancy in the literatures the interaction between the Berberine Chloride and DNA are of great significance. Nowadays, reverse micelles are the active field of scientific research because a tunable amount of water can be encapsulated within it which provides a membrane like environment.26-28 The amount of water or other polar solvents in the core of the reverse micelle can be varied. However, recently, several groups have replaced the polar solvents with hydrophilic ionic liquid in the core of the reverse micelle.29-30 Thus, the size of the reverse micelle is depended on the water (or other polar solvents) loading into the core of the reverse micelle which is denoted as and it can be defined as the ratio of the polar solvent molecules to surfactant molecules: 31 =

[ ] (1) [ ]

The water core of the reverse micelle have similar environment to that of biological systems. Thus, it provides an attractive model for biological systems. They mimic several properties and dynamics of the biological membranes avoiding the complexity associated with them. Thus, reverse micelle can be used as a tool to study the confinement effect and the dehydration of biological molecules.32-36 For example, DNA is a long, highly charged stiff polymers which must need to go through a condensation process in order to fit into the small tiny volume inside the cell and this process is known as DNA condensation. This condensation process has huge importance for the success of gene therapy protocols.37 Therefore, it is expected that after insertion of DNA into the nano-cavity of reverse micelle it turn into the condensed form where the size of the reverse micelle is comparable to that of DNA. A number of condensing agent such as charged or anionic polymers or electrolytes were reported in the literature till date. 38-42 Only a few literature reports are present where the core of the reverse micelle has been used as a condensing media for DNA.

40,43-46

In case of neutral reverse micelle, the spatial reconstruction

imposed by reverse micelle and the low dielectric constant of the polar core are main contributing factor for the DNA condensation process.40,44 However, for cationic and anionic reverse micelle, DNA structure is affected by the restricted water and the electrostatic interaction between the charged surface of RMs and the DNA.46,47 It is earlier reported that the factors which affect the DNA condensation process is related to the reorganization and the state of water



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

along the DNA chains48 and the compactness of the DNA in the nanopool of the reverse micelle is depended on the degrees of hydration also.49-50 In this present study, first we have explored the interaction between CT-DNA and Berberine Chloride (BBCl) in buffer via spectroscopic and circular dichroism measurements and we have observed a partial intercalation of BBCl into the planar base pair of DNA unlike to other classical intercalator such as Ethidium Bromide (EtBr). As a DNA condensing media, we have used anionic AOT (bis(2-ethyl hexyl)sulfosuccinate) and cationic BHDC (benzene dimethyl hexadecylammonium chloride) reverse micelle respectively. It is well established that the electrostatic interaction between the charged reverse micelle and DNA affect the DNA structure in the nanopool.49 So, we have preformed a comparative study of DNA condensation in two differently charged reverse micelles. We have used steady state and time resolved fluorescence techniques to determine the binding interaction between the Berberine Chloride and CT-DNA in the nanopool of the reverse micelle. However, the major problem associated with the fluorescence technique is massive interference of the Berberine chloride emission from the unbound BBCl in the reverse micelle without DNA. Thus, the background emission is unavoidable because the possibility of partioning to different microenvironments. For this reason, we have also performed the emission study of BBCl in reverse micelle in absence of DNA and we have compared the results with the presence of CT-DNA at a particular . Interplay of surface charge between RMs and DNA as well as the structural architecture of DNA in the nanopool mainly govern the interaction with BBCl. The interaction of the alkaloid with the RMs has been investigated to find a useful drug carrier in the physiological condition and the work also demonstrates how the nanopool of the differently charged RMs affects the interaction between BBCl and condensed DNA.

2. Experimental Section. 2.1. Materials: Berberine Chloride (BBCl) was obtained from Sigma-Aldrich. Dioctyl sulfosuccinate sodium salt (Na-AOT) and Benzyldimethylhexadecylammonium chloride (BHDC) were purchased from Sigma-Aldrich. Sodium salt of Deoxyribonucleic Acid (Calf Thymus DNA, CT DNA) and spectroscopic grade benzene was received from Sisco Research Chemical Laboratory (SRL), India. All the materials were used as received without any further 4 ACS Paragon Plus Environment

Page 4 of 37

Page 5 of 37

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


purification. Benzene was used as a non-polar solvent and a required amount of solid NaAOT and BHDC were added to benzene for the preparation of the reverse micelles and different amount of water was added to the reverse micelle to obtain the desired W0 values. Freshly prepared DNA solution was used in all the experiments. The homogeneous solution of CT-DNA was prepared by adding solid CT-DNA into the phosphate buffer of pH 7 and the solution was stored at 40C for 2 days with occasional shaking. The purity of DNA was verified by measuring the absorbance at 260 and 280 nm and the final concentration of the freshly prepared stock DNA solution is determined by measuring the absorbance at 260 nm using the molar absorbance coefficient ( ) of 13600 mol-1cm-1.51,52 The chemical structures of the all the above mentioned compounds are shown in scheme-1. 2.2. Instrumentation: Instrumentation section is discussed in Supporting Information. 3. Results and Discussion: 3.1. Mode of Interaction of CT-DNA with Berberine Chloride: Intercalation or Groove Binding? 3.1.1. Steady state UV-Vis and Fluorescence Studies: Berberine Chloride shows absorption maxima in water at 344 nm. However, with addition of different concentration of CT-DNA a large bathochromic shift is observed in the absorption spectra (figure 1(a)). It is generally accepted that, for intercalation mode of binding a large shift in absorption maxima is observed and the insignificant change in the absorption spectra signify the groove binding.51,53 The equilibrium binding is confirmed from the presence of isosbestic point which involve two systems comprising of free and bound Berberine Chloride and it also signifies the presence of one mode of binding. 53 BBCl shows very low fluorescence in buffer and the reported quantum yield of BBCl in D2O is (4.7 X 10-4).54 With gradual addition of CT-DNA, the fluorescence intensity of BBCl is significantly increased and a marked blue shift is observed in the emission spectra (figure 1(b)). Emission and absorption spectra are used to determine the binding constant (Kb) and the number of sites per nucleotide (n). The binding curves are plotted according to Scatchard method and analyzed by McGhill and Von-Hippel model55, which is defined as,



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

= (1 − ) "

#$%

#$(%$#)

&

(%$#)

Page 6 of 37

(2)

where, r is the ratio of concentration of bound drug and the total concentration of DNA nucleotides. '( is the free drug concentration. The concentration of bound drug and free drug is determined from the absorption and emission spectra. However, McGhill and Von-Hippel model for non-cooperative binding of ligand with N sites (where N is the ligand site size, i.e., the reverse of n) are not helpful to provide the adequate adjustment of and n to fit the binding isotherms obtained from absorption spectra and emission spectra. Thus, there is a possibility of existence of two independent binding sites and we have fitted the curves assuming a two site model (figure 2). The values of binding constant (kb1, kb2) are 6.7 X 104 M-1 and 1.09X106 M-1 respectively and n1 and n2 are obtained as 0.45 and 0.25 respectively. Angenot et al. have showed that Cryptolepine hydrochloride, an indoquinoline alkaloid intercalate into DNA which provides two independent non-cooperative binding sites.56 Thus, two sites model provide much better result and the values are comparable with the other intercalating agents such as ellipticines and acridines.57,58Thus, absorption and emission spectra of BBCl in presence of different concentration of CT-DNA gives us a preliminary idea about the binding interaction between them which can be further confirmed by the time resolved study. 3.1.2. Steady State Fluorescence Quenching Study and Effect of Ionic Strength: To further investigate the mode of binding between BBCl and CT-DNA fluorescence quenching study is performed and iodide ion is used as a quencher. The highly negative iodide ion is expected to be repelled in presence of negatively charged phosphate backbone of DNA molecule. In case of intercalation, small molecules are inserted into the planer bases of DNA. Thus, iodide molecules cannot quench the fluorescence of the molecules. However, in case of weak binding or groove binding, fluorescence of drug molecules is readily quenched in presence of iodide ion. The steady state fluorescence quenching of BBCl in presence of different concentration of NaI is followed using the well known Stern-Volmer equation, )* )

= 1 + ,- [.]

(3)

where, / and F are the fluorescence intensity of BBCl in absence and in presence of quencher, KI respectively, ,- is the Stern-Volmer constant and Q is the concentration of the quencher, KI. 6 ACS Paragon Plus Environment

Page 7 of 37

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


To compare the binding interaction between BBCl and CT-DNA we have used another popular DNA intercalating fluorophore, Ethidium-Bromide (EtBr). In presence of 0.4 (M) NaI, we have observed insignificant decrease in the fluorescence intensity of EtBr. However, a pronounced decrease in the fluorescence intensity occurred for the BBCl-DNA complex in presence of 0.25 (M) NaI (figure-3(a,b)). Thus, it clearly indicates that the protection efficiency of CT-DNA is much higher for EtBr compared to BBCl. However, the Stern-Volmer constant for BBCl in absence and in presence of CT-DNA in buffer is 26.8 M-1 and 6.27 M-1 respectively.25 Thus, the quenching value is decreased when BBCl was bound to CT-DNA which signifies the interaction between BBCl and DNA although the interaction is not so strong compared to classical DNA intercalator, EtBr. As BBCl carries positive charge and DNA contain negative charge on the phosphate backbone there is a possibility of electrostatic interaction between them. For this reason, we have performed the effect of ionic strength on BBCl-DNA binding. With increasing the ionic strength of the medium, the electrostatic interaction between the consecutive phosphate groups is screened. As a result, the helix of the DNA is shrunk due to the reduction in the unwinding tendency caused by the electrostatic repulsion present between the phosphate groups.59-61 Thus, with increasing the ionic strength of the medium, the electrostatic interaction between the positively charged fluorophore and DNA is reduced. With increasing concentration of NaCl, a strong electrolyte, the fluorescence intensity is decreased without any shift in the fluorescence maxima (figure 3(c)). This observation tells us that electrostatic interactions also play a role in the binding between DNA and BBCl. 3.1.3. Time Resolved Emission Spectra and Effect of Temperature: In order to get insight into the remarkable increase in the fluorescence intensity of BBCl after binding to CT-DNA we have performed time resolved measurement of BBCl in presence of different concentration of DNA. In buffer, the fluorescence decay of BBCl is very fast (98 ps) and it is close to our instrument response function and we have also detected one very weak long decay component of 4.09 ns which can be attributed to the solvated cluster of BBCl59 and it is confirmed by measuring the lifetime decays of BBCl in presence of different concentration of urea.62 Urea is well known as water structure breaker. However, there has been an extended debate on the influence of urea on the structure of water.63,64 Bakker et al.65 using mid infrared IR pump probe 7 ACS Paragon Plus Environment


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

spectroscopy showed that even at high concentration of urea (8M) the orientation dynamics of water is retained. However, a small amount of water is tightly associated with water forming specific water-urea complex.65 In this aspect, fluorescence lifetime of BBCl can be served as an indicator to explore the surroundings of the fluorophore. Now, with increasing concentration of urea the long component (4.09 ns) in the lifetime is reduced significantly (figure S1 and table S1 of Supporting Information) and the short component of 98 ps is increased slightly which may be due to the reduction in the non-radiative decay rate of BBCl in presence of urea. Urea can solvate the hydrophobic solute by replacing the water molecules from the hydration shell of the solutes.66 Therefore, urea rupture the hydrogen bonded cluster and correspondingly the long component of the lifetime is decreased with addition of urea. With addition of CT-DNA, the average lifetime of BBCl increases significantly (figure 4) and the emission decays can be satisfactory described by tri-exponential function with three distinct lifetime values (table 1). Multiexpoential lifetime decay of drug-DNA complexes is well reported in the literature.60,68 However, the exact location of the drug in the DNA is still uncertain. BBCl is highly water soluble and the fast component correspond to the unbound BBCl molecules and the other two components refer to the bound BBCl on the DNA surfaces. The third component (01 ) increases significantly with increasing concentration of CT-DNA (from 5.50 ns in presence of 0.040 mM CT-DNA to 8.21 ns in presence of 0.340 mM CT-DNA) which may be the outcome of intercalation of BBCl into the planar base pair of CT-DNA. The second component (02 ) has an intermediate lifetime which can be ascribed to other types of binding interaction, such as electrostatic interaction. Thus, the multiexponential lifetime also suggests two different types of binding interaction. Moreover, the gradual increase in the average lifetime of BBCl with addition of different concentration of CT-DNA strongly suggests the binding interaction between them. A much detail understanding of the increment of fluorescence of BBCl with addition of CTDNA has been conducted by separating the non-radiative rate constant (% ) from the radiative rate constant ( ). The radiative and non-radiative rate constant can be determined from the quantum yield and average lifetime using the following equations:


Page 8 of 37

Page 9 of 37

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


=

3456789

(4)

:;5

% = :;

#

5

−

(5)

The calculated value of radiative, non-radiative rate constant, quantum yield and average lifetime values are summarized in table S2 of Supporting Information. The non-radiative rate constants (% ) of BBCl are reduced reasonably after binding with CT-DNA. However, the change in the radiative rate constants ( ) remain imperceptible compared to % . Thus, the increase in the fluorescence lifetime or fluorescence intensity in presence of CT-DNA is the consequence of the reduction of the non-radiative decay path of BBCl. The different binding modes of CT-DNA can be perceived by performing temperature dependent emission study of BBCl-DNA complex. The emission intensity as well as the lifetime of the complex is decreased with gradual increase in the temperature (figure S2, Supporting Information) due to the increase in the non-radiative decay paths. The relative contribution of the long component which is attributed to the intercalation mode of binding remains fixed with increasing temperature (table S3 of Supporting Information). However, the relative contribution of the 2nd component (?2 ) is gradually decreasing and consequently, the contribution of the fast component (?# ) is increasing with increase in temperature. As intercalation mode involves strong binding between the BBCl and the planar base pair of CT-DNA ?1 remains unaltered. However, the other mode involves the weak interaction which is represented by the 2nd component in the lifetime value and such interaction become weak with increase in the temperature. We have increased the temperature from 298K to 343 K and it is confirmed from the CD measurement that the secondary structure of the CT-DNA remains unaltered upto 343 K which is discussed below. The decrease in the quantum yield with increase in the temperature is due to the increase in the non-radiative decays paths (figure 5(a)) and we have also determined the activation energy of the non-radiative decay process of BBCl in CT-DNA from the slope of the Arrhenius Plot of ln(% ) against 1/T as depicted in the inset of figure 5(a) and the activation energy thus obtained for the non-radiative decay of BBCl in CTDNA is -11.67kJ/mole. The binding interaction between CT-DNA and BBCl is further established from the steady state anisotropy measurement. Anisotropy measurements further help us to draw a conclusion about the rigidity of the surroundings of microenvironment provided by CT-DNA. We have observed a 9 ACS Paragon Plus Environment


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

Page 10 of 37

sharp increase in the fluorescence anisotropy of BBCl with addition of different concentration of CT-DNA (figure 5(b)). Thus, it is suggested that BBCl are relocated themselves into more restricted region compared to buffer and overall tumbling motion is observed irrespective of the nature of their binding. 3.1.4. Circular Dichroism (CD) Measurement: The conformational changes of the DNA molecule in presence of BBCl can be understood from the CD spectra. The far UV-CD spectra which shows a minimum at ~247 nm and a maximum at ~ 276 nm correspond to the right handed B form.60,69 The influence on the secondary structure of CT-DNA with increasing concentration of BBCl is followed by monitoring the far-UV spectra of CT-DNA as shown in figure 6. We have observed that the intensity of the 276 nm band is increased with increasing concentration of drug. The stacking contact between the base pair of the DNA is disrupted due to the intercalation of BBCl into the base pair of DNA (to accommodate the drug within the base pair).59,70,71However, groove binding or electrostatic interaction hardly affect the CD spectrum. Thus, this observation satisfactory establishes the intercalation of BBCl into CT-DNA. We have also performed temperature dependent CD measurement of BBCl/ CT-DNA and we have varied the temperature from 278K to 343K (figure S3, Supporting Information) and no significant change is observed in the CD spectra of CT-DNA which indicates that the secondary structure of CT-DNA remain unaltered upto this temperature and this also supports our conclusions drawn from the temperature dependent time resolved measurement discussed before. 3.1.5. Wobbling Motion of BBCl in CT-DNA: The rotational anisotropy of BBCl in presence of CT-DNA is defined as

() = (0)[?# expD−E0# F + ?2 expD−E02 F]

(6)

In the above equation, (0) is the initial anisotropy and the value is depended on the angle between absorption and emission dipole. ?# and ?2 are the relative contribution of 0# and 02 respectively and ?# is related to the generalized order parameter S by ?# = 2 which describes the degree of restriction on the wobbling in cone orientational motion. It is governed by the inequality, 1≥ 2 ≥0. Semicone angle, GH can be obtained from the order parameter by following the equation,


Page 11 of 37

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


2 = I0.5 (GH )(1 + GH )L2

(7)

The anisotropy decay of BBCl in presence of 0.34 mM of CT-DNA is shown in figure S4, Supporting Information and the decay is easily fitted by bi-exponential decay function. The slower component of rotational relaxation time of 2.93 ns (table 2) in CT-DNA may be due to the local tumbling of DNA segments.71-73 The biexponential decay can be rationalized that the probe molecule undergoing different kind of rotational motion in DNA such as (a) wobbling motion of the probe (b) translational motion of the probe along the surface of the DNA and (c) the overall motion of DNA.60 Due to the involvement of these kind of motions the anisotropy deviate from the single exponential decay. Now, one cannot expect the lateral diffusion of BBCl along the DNA-water interface because all the BBCl are bound to CT-DNA at their saturation level.74 Now, the faster relaxation time can be described as the motion of a restricted rotor having its transition dipole moment undergoing orientational diffusion within a semicone of angle (GH ) about an imaginary axis. We have calculated the semicone angle of BBCl in CT-DNA following equation 7 and we have found that the semicone angle observed for BBCl (23.40) is slightly larger than the semicone angle observed for EtBr intercalated into DNA (150).53 The hydrogen bonding interaction between the amino protons of the EtBr and the phosphate backbone may be the possible reason for such low GH observed for EtBr intercalated into DNA. However, the angle(GH ) is much lower compared to other groove binding probe such as DASPMI (420).53 Thus, all the studies suggest that BBCl intercalate into the planar base pair of CT-DNA. However, the mode of binding is slightly different from the classical intercalate probe, such as EtBr. We have mentioned earlier that Berberine Chloride is not a planar molecule and thus, it does not intercalate into the planar base pair of DNA by classical model. CPK space filling model suggests that the van der waals thickness of Berberine Chloride (4.5A0) is slightly larger than the thickness of Coralyne Chloride (3.4A0), a planar molecule which readily intercalate into the DNA helix.75 Wilson et al. have showed that Berebrine Chloride induced an increase in the viscosity of DNA, which is expected for the intercalation.75However, the increase in viscosity was not pronounced compared to other planar classic intercalator drug, such as quinacrine, coralyne etc and they have further demonstrated that the unwinding angle of plasmid DNA is less for BBCl compared to other intercalators which also suggests that BBCl persuades a different conformational changes in the DNA double helix on intercalation than classical 11 ACS Paragon Plus Environment


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

Page 12 of 37

intercalating molecules. Moreover, the first crystal structure of Berberine intercalated with a d(CGTACG)2 sequence was obtained by X-ray diffraction analysis and BBCl was found stacked in non-classial intercalation site formed by DNA duplexes.76 Thus, due to the partial saturation in the structure of BBCl, it partially intercalate into the base pair of CT-DNA which causes bending of the DNA helix and the electrostatic interaction also play a significant role in the binding between BBCl and CT-DNA. 3.2. BBCl in H2O/AOT/Benzene and H2O/BHDC/Benzene Reverse Micelles. 3.2.1. Steady State UV and Fluorescence Measurements: BBCl is a cationic probe molecule and among the different possible interactions electrostatic interaction (between a charged guest molecule and RMs) predominates in several dynamic phenomena inside the nanopool of charged RMs77 and to verify this we have chosen two oppositely charged RMs; AOT (negatively charged) and BHDC (positively charged). Figure 7 shows two absorption bands of BBCl at around 350 nm and 430 nm in AOT and BHDC RMs. The 350 nm band is assigned to strong M − M interaction. The absorption spectra are shifted towards longer wavelength with gradual addition of water into the core of the RMs (figure 7). However, in case of AOT RMs, due to the favorable electrostatic interaction between the negatively charged AOT and cationic Berberine there is a possibility of ion-dipole interaction and such types of interaction is not possible when BBCl is encapsulated into the cationic BHDC RMs. Beside these, BBCl is water soluble drug molecule. Thus, with increasing concentration of water in RMs we have observed hyperchromic shift in absorption spectra. Figure 8 shows the change in the fluorescence spectra of BBCl in two different RMs with different water contents. In both the cases, we have observed with increasing the water content emission maxima is red shifted and the intensity of the emission maxima is decreased. The red shift in the emission maxima signifies that the drug molecule is shifted towards the core of the RMs due to their high solubility in water.

BBCl shows very low fluorescence in water.

However, the fluorescence is significantly enhanced when BBCl is encapsulated into the RMs and such enhancement of emission intensity is also obtained with BBCl bound to surfactants78 and confined in the cavity of different guest molecules such as N cyclodextrin79, calixarenes80, cucurbiturils81 etc. With increasing the polarity of the medium the non-radiative transition of BBCl is increased and Inbaraj et al. proposed that such increment in the non-radiative transition 12 ACS Paragon Plus Environment

Page 13 of 37

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


is due to the significant hydrogen bonding interaction between the BBCl and polar solvents82. The shift in the emission maxima and the change in the fluorescence intensity of BBCl in presence of AOT and BHDC RMs in different values are plotted in figure 9(a, b) and the quantum yield (O) of BBCl in two RMs is plotted by varying the water content ( ) (figure 9(c)). The quantum yield of BBCl in RMs is much higher than in bulk solvents which signify the confinement of BBCl into the nanopool of the RMs affect the non-radiative decays of singlet excited state.83 The quantum yield of BBCl in 0.4 (M) AOT RMs (O = 0.13) is 1.5 times higher compared to BBCl in 0.4 (M) BHDC RMs (O = 0.085) at =0.00. The ion dipole interaction between the BBCl and AOT may be the possible reason for higher O value in AOT RMs and such kind of interaction is also reported between SDS and Berberine cation.84 The decrease in the quantum yield is much higher in case of BBCl entrapped in AOT reverse micelle compared to that of BHDC reverse micelle. The reactivity between two charged molecules is mainly governed by the electrostatic interaction i.e opposite charge attract each other while same charge repel each other. Thus, it is expected that positively charged Berberine is located at the watersurfactant interface due to the strong electrostatic interaction with the negatively charged AOT. With increasing the water content, BBCl is shifted towards the pool of AOT RMs and the emission intensity and the quantum yield is significantly reduced. However, in case of cationic BHDC RMs Berberine cation is expected to locate at the centre of the water pool of the reverse micelle to minimize the repulsive interaction. Thus, we have not observed any significant change in the quantum yield of BBCl with respect to compared to that of AOT RMs. 3.2.2. Time Resolved Fluorescence Studies. Time resolved fluorescence measurement provides better understanding about the local environment in which the fluorophores are localized. The time resolved decays of BBCl in two different reverse micelles are shown in figure 10 and the decays are successfully fitted with tri-exponential functions and the fitted lifetime values are shown in table 1. The non exponential decay of BBCl is due to the microheterogeneity of RMs. In general, the water molecules inside the reverse micelle can be broadly distinguished as bound water which are hydrogen bonded to the interface and the free water, located at the core of the RMs.85-87 However, FT-IR study reveals the possibility of four different kinds of water molecules in AOT reverse micelles namely free monomers, dimmers at the interface, monomer hydrogen bonded to interface and bulk water.88 In both the cases, with increasing the water content BBCl molecules are shifted towards the pool of the reverse micelle and the average 13 ACS Paragon Plus Environment


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

Page 14 of 37

lifetime of BBCl decreases significantly. The contribution of the short lifetime component increases with increasing the water content which signifies the relocation of BBCl from the interface to the pool of the RMs. However, the decrease is more prominent for AOT RMs as indicated by steady state results also. In presence of 0.4 (M) BHDC, there is a possibility of formation of ion pair (BB+Cl-) which significantly increase the non-radiative transition of BBCl.89However, the formation of ion pair hardly affect the absorption and emission spectra of BBCl in RMs. The formation of ion pair along with the Berberine cation in BHDC reverse micelle may also contribute to the multi-exponential decays. To get a vivid idea about the modulation of the excited state photophysics of BBCl upon encapsulation into the RMs we have calculated the radiative and non-radiative rate constants following equation 4 and 5 and the values are enlisted in table S2, Supporting Information. The non-radiative rate constant of BBCl in BHDC reverse micelle is much higher (4.42 X 108 s-1) compared to AOT reverse micelle (2.27 X 108 s-1). The formation of ion pair of BBCl in BHDC RMs accelerates the non-radiative decay processes. With addition of water, probe molecules are shifted towards the pool of the RMs, which significantly increase the non-radiative decay processes in AOT RMs. However, BBCl encapsulated in BHDC RMs, the nonradiative decay process does not change appreciably (figure S5, Supporting Information) which is expected from the steady state and time resolved results. 3.2.3. Time Resolved Fluorescence Anisotropy Measurements and Wobbling in Cone Model. Time resolved fluorescence anisotropy measurements were performed in order to understand the reorientation dynamics of BBCl in organized assemblies. Anisotropy decays of BBCl in AOT and BHDC RMs with varying are shown in figure 11 and the decays are adequately fitted with bi-exponential function following equation 6 and decay parameters are given in table 3. It is observed that both the slow and fast component is decreased with increasing the water content in the RMs which indicates that BBCl is relatively more free to move in large RMs. Thus, the average reorientation time (0RST ) of BBCl is decreased with increasing the water content in both the RMs and the 0RST of BBCl in 0.4 (M) AOT RMs at =0.00 is higher than that of BHDC RMs due to the preferential location of BBCl at micellewater interface in AOT RMs. The water droplets in the RMs are spherical in nature and the


Page 15 of 37

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


overall rotation of the RMs can be calculated by following the Stokes-Einstein-Debye (SED) relation: 0U =

VWXY Z

(8)

1[\ ]

where, ^ is the hydrodynamic radius of the reverse micelle. _ is the viscosity of the medium and ` and T are the Boltzmann constant and temperature respectively. The hydrodynamic radius of the reverse micelles is obtained from the earlier literatures77 and they correlate well with the values obtained from DLS measurement. The viscosity of the medium can be calculated by following the equation:90 _abcdefb% = _abcSg%e (1 + [η]')

(9)

In this equation, _abcSg%e is the viscosity of benzene and [η] is the intrinsic viscosity which is 3.01 cm3g-1 for AOT in n-heptane at 298K and we have assumed that the value is same in benzene also. The value of _abcdefb% we obtained in AOT RMs is close enough to the experimentally measured value and _abcdefb% in BHDC RMs was obtained by measuring the viscosity of the solution. Thus, the 0U values are obtained enlisted in table 4 and 5 and with increasing the water content the value is considerably increased due to the increase in the hydrodynamic radius of each of the RMs. 0# which correspond to the slower component of the rotational motion are significantly lower than the micellar rotation in both the RMs. Moreover, 0# value is gradually decreased with increasing the water content. Based on this fact, we can conclude that the 0#does not correspond to the overall micellar rotation. Thus, both 0# and 02 each represent a combination, rather than individual diffusion processes and the restricted rotation in the RMs can be explained by two steps model. In this model, fluorophores which are located at the water-micellar interface experiences a lateral diffusion (0 ) inside or out of the curved surfaces and the shorter component in the rotational relaxation represent the motion of a restricted rotor where the transition dipole moment can undergo an orientational relaxation within a cone of semiangle G.91The analytical parameters which were obtained from the anisotropy measurements are summarized in table 4 and 5 and the useful equations are given in the Supporting Information.



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

Page 16 of 37

With increasing the water content in the RMs, the compactness of water-micellar interface is gradually decreased and the lateral diffusion time becomes faster and the corresponding diffusion coefficient (ij ) is increased. However, the increase in the ij is much higher in case of AOT RMs compared to BHDC RMs (figure S6, Supporting Information). This is due to the electrostatic interaction, BBCl are prone to locate at the water–micellar interface in AOT RMs at lower W0. Thus, with increasing the , probe molecules are rearranged themselves towards the pool of the RMs and thus the compactness sensed by the fluorophores are reduced drastically. However, in case of BHDC RMs, being cationic in nature BBCl is away from the positively charged micellar surface at low water content and hence with increase the water content the change in the lateral diffusion coefficient in minimal compared to AOT RMs.

The order

parameter values are also gradually decreased with addition of water which also indicates the relocation of probe molecule to the nanopool of the reverse micelle from the micellar interface as the values of S range from 0 (unrestricted motion) to 1 (restricted motion). 3.3. CT-DNA in Reverse Micelle and interaction with BBCl: The sizes of the RMs upon encapsulation of CT-DNA were measured using DLS instrument. In absence of any DNA the size of the RMs are gradually increased proportionally with increasing the water content. However, in presence of different concentration of CT-DNA two types of reverse micelles are observed form DLS measurements which are empty RMs and DNA containing large RMs (figure S7 of Supporting Information). In this aspect, it is important to mention that the RMs containing DNA may not be spherical in nature and thus the size of the RMs determined from DLS may not be accurate. Large RMs containing DNA is also reported in the literature.40,49,92 However, Luisi et al proposed that very large size of reverse micelles can be attributed to the cluster of small reverse micelles.92 The water pool of the RMs is extremely rich in negative or positive charges depending on the polar head groups of the surfactants. Thus, when the water soluble DNA with highly negative phosphate backbone is added to the RMs it is expected that DNA molecule is located at the water pool of the RMs in case of AOT reverse micelle and in case of cationic BHDC RMs it is located at the water-micellar interface and it is plausible that DNA may transform into its condense form. For this reason, we have also performed CD measurement. However, the problem associated with benzene as a nonpolar solvent is reflected in the large background in CD spectrum. So, we are unable to detect any 16 ACS Paragon Plus Environment

Page 17 of 37

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


resolvable signal in the CD spectrum in the range between 200-270 nm. However, partial information can be gained by using Ethidium Bromide (EtBr), a classic intercalator. Intercalation of EtBr into the planar base pair significantly affect the CD spectrum of CT-DNA and an induce CD spectra is generated at 308 nm and a shoulder band at 330 nm (figure S8 (a) of Supporting Information).93 Earlier, we have observed that BBCl partially intercalate into the CT-DNA. But, it exhibits very weak induced CD (ICD) spectrum (inset of figure 6). ICD is primarily depended on the angular orientation of the intercalator and moderately depend on the location of intercalation site.94Thus, the DNA condensation process can be monitored through the change in the ICD spectrum of EtBr/CT-DNA complex (CDNA: CEtBr =5:1). In case of AOT RMs, we do not observe any ICD spectrum in presence of EtBr. However, we have observed a remarkable red shift in the lmRn of the CD spectrum with respect to normal buffer solution (figure S8(b) of Supporting Information) and in case of BHDC RMs, the ICD spectrum is remained intact and it is associated with the 270 nm band (tail part is showed in the figure S8 (c) of Supporting Information) which is observed for CT-DNA in only buffer solution. This appreciable absorption at longer wavelength is the characteristics of o spectra

49,92,95

and DNA in RMs undergoes a

transition from B form to o form which is the clear signature of condense form of DNA. We have observed thread like morphology along with some gluobular aggregate of DNA encapsulated in RMs in FLIM images and FESEM images (figure 12). Similar types of aggregate are also reported in the literature.40 However, the structural transition of DNA from super coiled structure to other forms is mainly governed by the charge of the surfactants or other additives such as dendrimers. Nylander et al have showed that DNA can be transformed into globular aggregates, rod or toroid depending on the size of the dendrimers.96 The interaction between BBCl and CT-DNA in the nanopool of the RMs is mainly governed by the electrostatic interaction. With addition of 0.5 mM CT-DNA in AOT RMs (W0 = 4.00), we have not observed any shift in the emission maxima of BBCl (figure S9 of Supporting Information) and accordingly, negligible change in the fluorescence lifetime of BBCl is observed which promptly tell us that the insignificant interaction between the BBCl and CT-DNA in the nanopool of the AOT RMs (figure S10 of Supporting Information). Now, we have already showed that a significant amount of fluorophores are located at the water-micellar interface in AOT RMs and due to the highly negative phosphate backbone DNA molecules prefer to stay at the core of the RMs. Beside these, we have observed that with increasing the water content in the 17 ACS Paragon Plus Environment


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

Page 18 of 37

AOT RMs probe molecules are shifted towards the pool which is confirmed from the fluorescence lifetime as well as the rotational relaxation study. Due to the condensation of DNA in the nanopool of the RMs the preferential binding site of CT-DNA may not be exposed to BBCl molecules. Pal et al have also demonstrated that the efficiency of intercalation of EtBr molecule is decreased in AOT reverse micelle due to the decrease in the DNA curvature.49,97 However, a significant blue shift is observed in the emission spectra of BBCl when CT-DNA is encapsulated in the BHDC RMs (figure S9 of Supporting Information) and RMs containing BBCl-DNA shows a long decay component of 11 ns in the fluorescence transient (figure 13 (a)) and this long component indicates the intercalation of BBCl molecules into the planar base pair of DNA. Because, in buffer solution also similar component is observed and we have also observed increase in the rotational relaxation time of BBCl in DNA encapsulated BHDC RMs. The average rotational relaxation time of BBCl is increased from 0.73 ns in absence of DNA containing reverse micelle ( = 4) to 4.23 ns in DNA containing RMs ( = 4) which shows the tumbling motion of BBCl molecules (figure 13(b)). Now, we have mentioned earlier that BBCl molecules are located at the pool of the BHDC RMs to minimize the electrostatic repulsion between the BB+ and cationic BHDC surfactants. However, when highly negative DNA molecules are added to the RMs solution the phosphate backbone of DNA strongly interact with the BHDC and the probe molecules are relocated themselves to the DNA surface. More interestingly, the contribution to the long decay component of BBCl in DNA containing BHDC RMs and buffer remains almost same which signify that BBCl readily intercalate into the DNA base pair in BHDC RMs contrary to that of AOT RMs. Beside the electrostatic interaction in the nanopool of the RMs the structural architecture of condensed DNA in the nanopool of the RMs may also contribute to such difference which is observed in AOT and BHDC RMs. 4. Conclusion: In conclusion, the present study depicted the binding interaction between Berberine Chloride and CT-DNA in buffer solution as well as in the aqueous nanopool of AOT and BHDC RMs. Albeit the literature debate regarding the binding mechanism between CTDNA and BBCl we have proposed that BBCl partially intercalate into the base pair of DNA and the nonplanar segment of the molecule is projected towards the groove and interacted via electrostatic attraction. Moreover, this study provide detailed information regarding the dynamics and location of this alkaloid in two differently charged reverse micelles and it is established that the location as well as the dynamics of BBCl is principally governed by the electrostatic 18 ACS Paragon Plus Environment

Page 19 of 37

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


interaction between the charged surfactant and BBCl. Wobbling in cone model indicates the tumbling motion of BBCl after binding to CT-DNA and the calculated cone angle is close enough to EtBr intercalated CT-DNA and among the RMs, the larger semicone angle is observed for the BHDC RMs which is due to the repulsive interaction between the cationic BHDC and positive BBCl molecules which pushes the probe molecules towards the water pool of the RMs. The CD spectra of CT-DNA indicate the condensed form of DNA after encapsulation into the RMs and the binding interaction between BBCl and CT-DNA is significantly altered in both the RMs which is mainly due to the electrostatic interaction of DNA with the charged surfactants and the condensation of DNA in the nanopool of the RMs. Thus, these experimental evidences provide further insight into the mechanism of DNA condensation in the pool of RMs. Acknowledgment: N.S. gratefully acknowledges SERB, Department of Science and Technology (DST) and Council of Scientific and Industrial Research (CSIR), Government of India for providing generous research grant. A.R. is thankful to CSIR, N. K., D. B. are thankful to IIT Kharagpur, for providing their research fellowships. Supporting Information: Instrumentation, Calculation for Wobbling Cone model, table of Fluorescence quantum yield, radiative and nonradiative decays in different assemblies, Fluorescence lifetime in presence of Urea, Temperature dependent fluorescence lifetime and CD spectra of BBCl bound to CT-DNA, plot of non-radiative rate constants in different RMs, Plot of lateral diffusion time of BBCl in RMs, DLS intensity plot of CT-DNA encapsulated in RMs, CD spectra of CT-DNA in RMs, Normalized Fluorescence Spectra of BBCl in DNA encapsulated RMs and the fluorescence lifetime plot of BBCl in DNA encapsulated RMs are shown in the Supporting Information. This information is available free of charge via the Internet at http://pubs.acs.org.

Reference: (1) Maung U, K.; Khin, M.; Wai, N. N.; Kyaw, A.; Tin, U. Clinical Trial of Berberine in Acute Watery Diarrhea. Br Med J, 1985, 291, 1601-1605. (2) Domingo, M. P.; Pardo, J.; Cebolla, V.; Gálvez, E. Berberine: A Fluorescent Alkaloid with a Variety of Applications from Medicine to Chemistry. Mini-Rev. Org. Chem. 2010, 7, 335−340. (3) Giri, P.; Kumar, G. S. Molecular Recognition of Poly(A) Targeting by Protoberberinealkaloids: In Vitro Biophysical Studies and Biological Perspectives. Mol. BioSyst. 2010, 6, 81−88.



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

Page 20 of 37

(4) Sinha, R.; Kumar, G. S. J. Interaction of Isoquinoline Alkaloids with an RNA Triplex: Structural and Thermodynamic Studies of Berberine, Palmatine, and Coralyne Binding to Poly(U).Poly(A)*Poly(U). J. Phys. Chem. B 2009, 113, 13410−13420. (5) Islam, M. M.; Kumar, G. S. RNA-Binding Potential of Protoberberine Alkaloids: Spectroscopic and Calorimetric Studies on the Binding of Berberine, Palmatine, and Coralyne to Protonated RNA Structures. DNA and Cell Biol. 2009, 28, 637−650. (6) Hu, Y. J.; Liu, Y.; Xiao, X. H. Investigation of the Interaction between Berberine and Human Serum Albumin. Biomacromolecules 2009, 10, 517−521. (7) Liu, Y. F.; Yu, H. M.; Zhang, C.; Cheng, Y. F.; Hu, L. K.; Meng, X. H.; Zhao, Y. X. Protective Effects of Berberine on Radiation-Induced Lung Injury Via Intercellular Adhesion Molecular-1 and Transforming Growth Factor-Beta-1 in Patients With Lung Cancer. Eur. J. Cancer 2008, 16, 2425−2432. (8) Domadia, P. N.; Bhunia, A.; Sivaraman, J.; Swarup, S.; Dasgupta, D. Berberine Targets Assembly of Escherichia Coli Cell Division Protein FtsZ. Biochemistry 2008, 47, 3225−3234. (9) Jantova, S.; Cipak, L.; Cernakova, M.; Kostalova, D. Effect of Berberine on Proliferation, Cell Cycle and Apoptosis in HeLa and L1210 cells. J. Pharm. Pharmacol. 2003, 55, 1143−1149. (10) Asai, M.; Nobuhisa, I.; Yoshikawa, A.; Aizaki, Y.; Ishiura, S.; Saido, T. C.; Maruyama, K. Berberine Alters the Processing of Alzheimer’s Amyloid Precursor Protein to Decrease Aβ Secretion. Biochem. Biophys. Res. Commun. 2007, 352, 498−502. (11) Kong, W.; Wei, J.; Abidi, P.; Lin, M.; Inaba, S.; Li, C.; Wang, Y.;Wang, Z.; Si, S.; Pan, H. et al. Berberine is a Novel Cholesterol-Lowering Drug Working Through a Unique Mechanism Distinct from Statins. Nat. Med. 2004, 10, 1344−1351. (12) Hahn, F. E.; Ciak, J. Ann. Elimination of Bacterial Episomes by DNA-Complexing Compounds. N. Y. Acad. Sci. 1971 182,295-304. (13) Hahn, F.E.; Cuak, J. Berberine. In: Corcoran JW, Hahn ed. Antibiotics. New York: Springer-Verlag, 1975, 577–584. (14) Li, T.-K. E.; Bathory, E.; La Voie, E. J.; Srinivasan, A. R.; Olson, W. K.; Sauers, R. R.; Liu, L. F.; Pilch, D. S. Human Topoisomerase I Poisoning by Protoberberines: Potential Roles for Both Drug−DNA and Drug−Enzyme Interactions. Biochemistry 2000, 39, 7107. (15) Long, B. H.; Balasubramanian, B. N. Non-Camptothecin Topoisomerase I Active Compounds as Potential Anticancer Agents. Exp. Opin. Ther. Pat. 2000, 10, 635. (16) Shi, Y.; Guo, C.; Sun, Y.; Liu, Z.; Xu, F.; Zhang, Y.; Wen, Z.; Li, Z. Interaction between DNA and Microcystin-LR Studied by Spectra Analysis and Atomic Force Microscopy. Biomacromolecules 2011, 12, 797−803. (17) Elder, R. M.; Emrick, T.; Jayaraman, A. Understanding the Effect of Polylysine Architecture on DNA Binding Using Molecular Dynamics Simulations. Biomacromolecules 2011,12, 3870−3879. 20 ACS Paragon Plus Environment

Page 21 of 37

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


(18) Akiyama, Y.; Ma, Q.; Edgar, E.; Laikhter, A.; Hecht, S. M. Identification of Strong DNA Binding Motifs for Bleomycin. J. Am.Chem. Soc. 2008, 130, 9650−9651. (19) Campbell, N. H.; Smith, D. L.; Reszka, A. P.; Neidle, S.; O’Hagan, D. Fluorine in Medicinal Chemistry: β-fluorination of Peripheral Pyrrolidines Attached to Acridine Ligands Affects Their Interactions with G-quadruplex DNA. Org. Biomol. Chem. 2011, 9, 1328−1331. (20) Lyles, M. B.; Cameron, I. L. Interactions of the DNA Intercalator Acridine Orange, with Itself, with Caffeine, and with Double Stranded DNA. Biophys. Chem. 2002, 96, 53−76. (21) Wilson, W. D.; Gough, A. N.; Doyle, J. J.; Davidson, M. W. Coralyne. Intercalation with DNA as a Possible Mechanism of Antileukemic Action J. Med. Chem. 1976, 19, 1261-1263 (22) Krey, A. K.; Hahn, F. E. Berberine: Complex with DNA. Science , 1969, 166, 755-757 (23) Li, W.-Y.; Lu, H.; Xu, C.-X.; Zhang, J.-B.; Lu, Z.-H. Spectroscopic and Binding Properties of Berberine to DNA and Its Application to DNA Detection . Spectrosc. Lett. 1998, 31, 12871298. (24) Saran, A.; Srivastava, S.; Coutinho, E.; Maiti, M. 1H NMR Investigation of the Interaction of Berberine and Sanguinarine with DNA. Indian J. Biochem. Biophys. 1995, 32, 74-77. (25) Li, X.L.; Hu, Y.J.; Wang, H.; Yu, B.Q.; Yue, H.L. Molecular Spectroscopy Evidence of Berberine Binding to DNA: Comparative Binding and Thermodynamic Profile of Intercalation. Biomacromolecules 2012, 13, 873−880. (26) Levinger, N. E. Water in Confinement. Science 2002, 298, 1722−1723. (27) Nandi, N.; Bhattacharyya, K.; Bagchi, B. Dielectric Relaxation and Solvation Dynamics of Water in Complex Chemical and Biological Systems. Chem. Rev. 2000, 100, 2013-2045. (28) Bhattacharyya, K.; Bagchi, B. Slow Dynamics of Constrained Water in Complex Geometries. J. Phys. Chem. A 2000, 104, 10603-10613. (29) Gao, H. X.; Li, J. C.; Han, B. X.; Chen, W. N.; Zhang, J. L.; Zhang, R.; Yan, D. D. Microemulsions With Ionic Liquid Polar Domains. Phys. Chem. Chem. Phys. 2004, 6, 2914−2916. (30) Eastoe, S.; Gold, S. E.; Rogers, A.; Paul, T.; Welton, R. K.; Heenan, I. G. Ionic Liquid-inOil Microemulsions. J. Am. Chem. Soc. 2005, 127, 7302−7303. (31) Hauser, H.; Haering, G.; Pande, A.; Luisi, P. Interaction of Water with Sodium Bis(2-ethyl1-hexyl) Sulfosuccinate in Reversed Micelles). J. Phys. Chem. 1989, 93, 7869−7876. (32) Mukherjee, S.; Chowdhury, P.; Gai, F. Tuning the Cooperativity of the Helix-Coil Transition by Aqueous Reverse Micelles. J. Phys. Chem. B 2006, 110, 11615−11619. (33) Mukherjee, S.; Chowdhury, P.; Gai, F. Effect of Dehydration on the Aggregation Kinetics of Two Amyloid Peptides. J. Phys. Chem. B 2009, 113, 531−535.



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

Page 22 of 37

(34) Martinez, A. V.; DeSensi, S. C.; Dominguez, L.; Rivera, E.; Straub, J. E. Protein Folding in a Reverse Micelle Environment: The Role of Confinement and Dehydration. J. Chem. Phys. 2011, 134, 055107. (35) Rakshit, S.; Saha, R.; Pal, S.K. Modulation of Environmental Dynamics at the Active Site and Activity of an Enzyme under Nanoscopic Confinement: Subtilisin Carlsberg in Anionic AOT Reverse Micelle. J. Phys. Chem. B 2013, 117, 11565−11574. (36) Banerjee, D.; Sinha, S.S.; Pal, S.K. Interplay between Hydration and Electrostatic Attraction in Ligand Binding: Direct Observation of Hydration Barrier at Reverse Micellar Interface. J. Phys. Chem. B 2007, 111, 14239-14243. (37) Demeneix, B.; Hassani, Z.; Behr. J.P. Towards Multifunctional Synthetic Vectors. Curr. Gene Ther., 2004, 4, 445–455. (38) Bloomfield, V. A. Condensation of DNA by Multivalent Cations: Considerations on Mechanism. Biopolymers 1991, 31, 1471–1481. (39) Bloomfield, V. A. DNA Condensation. Curr Opin Struct Biol 1996, 6, 334–341. (40) Budker, V. G.; Slattum, P. M.; Monahan, S. D.; Wolff, J. A. Entrapment and Condensation of DNA in Neutral Reverse Micelles. Biophys J. 2002, 82, 1570–1579. (41) Strzelecka, T. E.; Rill, R. L. A 23Na-NMR Study of Sodium–DNA Interactions in Concentrated DNA Solutions at Low-Supporting Electrolyte Concentration. Biopolymers 1990, 30, 803–814. (42) Strzelecka, T. E.; Rill, R. L. Phase Transitions of Concentrated DNA Solutions in Low Concentrations of 1 : 1 Supporting Electrolyte. Biopolymers 1990, 30, 57–71. (43) Osfouri, S.; Stano. P.; Luisi, P.L. Condensed DNA in Lipid Microcompartments. J. Phys. Chem. B, 2005, 109, 19929-19935. (44) Barone, G.; Longo, A.; Ruggirello, A.; Silvestri, A.; Terenzia, A.; Liveri, V.T. Confinement Effects on the Interaction of Native DNA with Cu(II)–5-(triethylammoniummethyl)Salicylidene ortho-Phenylendiiminate in C12E4 Liquid Crystals. Dalton Trans., 2008, 4172–4178. (45) Airoldi, M.; Boicelli, C. A.; Gennaro, G.; Giomini, M.; Giuliani, A.M.; Giustinic. M. A Spectroscopic Study of Poly(dA-dT). Poly(dA-dT) Inmicroemulsions. Phys. Chem. Chem. Phys., 2000, 2, 4636È4641. (46) Airoldi, M.; Boicelli, C.A.; Gennaro,G.; Giomini, M.; Giuliani, A.M.; Giustinic, M.A.; Scibetta. L. Different Factors Affecting PolyAT Conformation in Microemulsions: Effects of Variable P0 and KCl Concentration. Phys. Chem. Chem. Phys., 2002, 4, 3859–3864 (47) Murphy, E.A.; Waring, A.J.; Haynes, S.M.; Longmuir, K.J. Compaction of DNA in an Anionic Micelle Environment Followed by Assembly into Phosphatidycholine Liposomes. Nucleic Acids Res.,2000, 28, 2986-2992.


Page 23 of 37

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


(48) Reich, Z.; Ghirlando, R.; Minsky, A. Secondary Conformational Polymorphism of Nucleic Acids as a Possible Functional Link between Cellular Parameters and DNA Packaging Processes. Biochemistry 1991, 30 (31), 7828-7836. (49) Sarkar, R.; Pal, S.K. Ligand–DNA Interaction in a Nanocage of Reverse Micelle. Biopolymers 2006, 83(6), 675-686. (50) Shaw, A. K.; Sarkar, R.; Pal, S. K. Direct Observation of DNA Condensation in a NanoCage by Using a Molecular Ruler. Chem Phys Lett 2005, 408, 366–370. (51) Mati, S.S.; Roy, S.S.; Chall, S.; Bhattacharya, S.; Bhattacharya, S.C.; Unveiling the Groove Binding Mechanism of a Biocompatible Naphthalimide-Based Organoselenocyanate with Calf Thymus DNA: An “Ex Vivo” Fluorescence Imaging Application Appended by Biophysical Experiments and Molecular Docking Simulations. J. Phys. Chem. B 2013, 117, 14655−14665. (52) Ghosh, S.; Banik, D.; Roy, A.; Kundu, N.; Kuchlyan J.; Sarkar, N. Spectroscopic Investigation of Binding Interaction of a Membrane Potential Molecule in Various Supramolecular Confined Environments: Contrast Behavior of Surfactant Molecules to Relocate or Release of Probe between Nanocarriers and DNA Surface. Phys. Chem. Chem. Phys., 2014,16, 25024-25038. (53) Sahoo, D.; Bhattacharya, P.; Chakravorti, S. Quest for Mode of Binding of 2-(4(Dimethylamino)styryl)-1-Methylpyridinium Iodide with Calf Thymus DNA. J. Phys. Chem. B 2010, 114, 2044−2050. (54) Inbaraj, J. J.; Kukielczak, B. M.; Bilski, P.; Sandvik, S. L.; Chignell, C. F. Photochemistry and Photocytotoxicity of Alkaloids from Goldenseal (Hydrastis canadensis L.) 1. Berberine. Chem. Res. Toxicol. 2001, 14, 1529. (55) McGhee, J.D.; Hippel, P,H,V. Theoretical Aspects of DNA-Protein Interactions : Cooperative and Non-co-operative Binding of Large Ligands to a One-dimensional Homogeneous Lattice. J. Mol. Biol. 1974, 86, 469-489. (56) Bonjean, K.; De Pauw-Gillet, M.C.D.; Defresne, M.P.; Colson, P.; Houssier, C.; Dassonneville, L.; Bailly, C.; Greimers, R.; Wright, C.; Quetin-Leclercq, J.; Tits, M.; Angenot, L. The DNA Intercalating Alkaloid Cryptolepine Interferes with Topoisomerase II and Inhibits Primarily DNA Synthesis in B16 Melanoma Cells. Biochemistry 1998, 37, 5136-5146 (57) Kohn, K. W., Waring, M. J., Glaubiger, D., Friedman, C.A. Intercalative Binding of Ellipticine to DNA. Cancer Res. 1975, 35, 71-76. (58) Wilson, W. R., Baguley, B. C., Wakelin, L. P. G., Waring, M. J. Interaction of the Antitumor Drug 4'-(9-Acridinylamino)methanesulfon-m-anisidide and Related Acridines with Nucleic Acids. Mol. Pharmacol.1981 20, 404-414. (59) Ganguly, A.; Ghosh, S.; Guchhait, N. Spectroscopic and Viscometric Elucidation of the Interaction between a Potential Chloride Channel Blocker and Calf-Thymus DNA: the Effect of Medium Ionic Strength on the Binding Mode. Phys. Chem. Chem. Phys., 2015, 17, 483—492. 23 ACS Paragon Plus Environment


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

Page 24 of 37

(60) Paul, B.K.; Guchhait, N. Exploring the Strength, Mode, Dynamics, and Kinetics of Binding Interaction of a Cationic Biological Photosensitizer with DNA: Implication on Dissociation of the Drug–DNA Complex via Detergent Sequestration. J. Phys. Chem. B, 2011, 115, 11938– 11949. (61) Modukuru, N. K.; Snow, K. J.; Perrin, B. S.; Kumar, C. V. Contributions of a Long Side Chain to the Binding Affinity of an Anthracene Derivative to DNA. J. Phys. Chem. B 2005, 109, 11810–11818 (62) Ganguly, A.; Jana, S.; Ghosh, S.; Dalapati, S.;.Guchhait, N. Solvent Modulated Photophysics of 9-Methyl Anthroate: Exploring the Effect of Polarity and Hydrogen Bonding on the Emissive State. Spectrochim. Acta, Part A, 2013, 112, 237–244. (63) Bandyopadhyay, D.; Mohan, S.; Ghosh, S.K.; Choudhury, N. Molecular Dynamics Simulation of Aqueous Urea Solution: Is Urea a Structure Breaker? J. Phys. Chem. B 2014, 118, 11757−11768 (64) Funkner, S.; Havenith, M.; Schwaab, G. Urea, a Structure Breaker? Answers from THz Absorption Spectroscopy J. Phys. Chem. B 2012, 116, 13374−13380. (65) Rezus, Y.L.A.; Bakker, H.J. Effect of Urea on the Structural Dynamics of Water. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 18417−18420. (66) Breslow, R.; Guo, T. Surface Tension Measurements Show that Chaotropic Salting-in Denaturants are not Just Water-structure Breakers. Proc. Natl. Acad. Sci. USA 1990, 87, 167– 169.; (67) Breslow, R. Hydrophobic Effects on Simple Organic Reactions in Water. Acc. Chem. Res, 1991, 24, 159–164 (68) Saha, I.; Hossain, M.; Kumar, G.S. Sequence-Selective Binding of Phenazinium Dyes Phenosafranin and Safranin O to Guanine−Cytosine Deoxyribopolynucleotides: Spectroscopic and Thermodynamic Studies. J. Phys. Chem. B, 2010, 114, 15278–15287. (69) Garbett, N.C.; Ragazzon, P.A. ;. Chaires, J.B. Circular dichroism to Determine Binding Mode and Affinity of Ligand-DNA Interactions. Nat. Protoc., 2007, 2, 3166–3172. (70) Ganguly, A.; Paul, B.K.; Ghosh, S.; Dalapati, S.; Guchhait, N. Interaction of a Potential Chloride Channel Blocker with a Model Transport Protein: a Spectroscopic and Molecular Docking Investigation. Phys. Chem. Chem. Phys., 2014, 16, 8465–8475. (71) A. Jha, J. B. Udgaonkar and G. Krishnamoorthy, Characterization of the Heterogeneity and Specificity of Interpolypeptide Interactions in Amyloid Protofibrils by Measurement of SiteSpecific Fluorescence Anisotropy Decay Kinetics J. Mol. Biol., 2009, 393, 735–752.


Page 25 of 37

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


(72) Ranjit, S.; Gurunathan, K.; Levitus, M. Photophysics of Backbone Fluorescent DNA Modifications: Reducing Uncertainties in FRET. J. Phys. Chem. B 2009, 113, 7861–7866 (73) Ramreddy, T.; Rao, B. J.; Krishnamoorthy, G. Site-Specific Dynamics of Strands in ss- and dsDNA As Revealed by Time-Domain Fluorescence of 2-Aminopurine. J. Phys. Chem. B 2007, 111, 5757 (74) Shaw, A.K.; Pal, S.K. Fluorescence Relaxation Dynamics of Acridine Orange in Nanosized Micellar Systems and DNA. J. Phys. Chem. B 2007, 111, 4189-4199. (75) Davidson, M. W.; Lopp, I.; Alexander, S.; Wilson, W.D. The Interaction of Plant Alkaloids with DNA. II. Berberinium Chloride. Nucleic Acids Res., 1977,4, 2697-2712. (76) Ferraroni, M.; Bazzicalupi, C.; Biliab, A.R.; Gratteri, P. X-Ray diffraction Analyses of the Natural Isoquinoline Alkaloids Berberine and Sanguinarine Complexed with Double Helix DNA d(CGTACG). Chem. Commun., 2011, 47, 4917–4919 4917. (77) Singh, P.K.; Kumbhakar, M.; Pal, H.; Nath, S. A Nano-Confined Charged Layer Defies the Principle of Electrostatic Interaction. Chem. Commun., 2011,47, 6912-6914. (78) Megyesi, M.; Biczok, L. Berberine Alkaloid as a Sensitive Fluorescence Probe for Bile Salt Aggregates, J. Phys. Chem. B, 2007, 111, 5635– 5639. (79) Yu, J.S.; Wei, F.D.; Gao, W.; Zhao, C.C. Thermodynamic Study on the Effects of Cyclodextrin Inclusion with Berberine, Spectrochim. Acta, Part A, 2002, 58, 249–256. (80) Megyesi, M.; Biczok, L. Considerable Fluorescence Enhancement upon Supramolecular Complex Formation between Berberine and p-Sulfonated Calixarenes, Chem. Phys. Lett., 2006, 424, 71–76. (81) Megyesi, M.; Biczok L.; Jablonksi, I. Highly Sensitive Fluorescence Response to Inclusion Complex Formation of Berberine Alkaloid with Cucurbit[7]uril, J. Phys. Chem. C, 2008, 112, 3410–3416. (82) Inbaraj, J.J.; Kukielczak, B.M.; Bilski, P.; Sandvik, S.L.; Chignell, C.F. Photochemistry and Photocytotoxicity of Alkaloids from Goldenseal (Hydrastis canadensis L.) 1. Berberine, Chem. Res. Toxicol., 2001, 14, 1529–1534. (83) Diaz, M.S.; Freile, M.L.; Guti´errez, M.I. Solvent Effect on the UV/Vis Absorption and Fluorescence Spectroscopic Properties of Berberine. Photochem. Photobiol. Sci., 2009, 8, 970– 974. (84) Iwunze, M.O. Media Infuence on the Enhancement of the Fluorescence of Berberine Hydrochloride. Monatsh. Chem., 2000, 131, 429–435.



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

(85) Verma, P.K.; Saha, R.; Mitra, R.K.; Pal, S.K. Slow Water Dynamics at the Surface of Macromolecular Assemblies of Different Morphologies. Soft Matter, 2010, 6, 5971–5979. (86) Verma, P.K.; Makhal, A.; Mitra, R.K.; Pal, S.K. Role of Solvation Dynamics in the Kinetics of Solvolysis Reactions in Microreactors. Phys. Chem. Chem. Phys., 2009, 11, 8467–8476. (87) Hazra, P; Chakrabarty, D; Sarkar, N. Intramolecular Charge Transfer and Solvation Dynamics of Coumarin 152 in Aerosol-OT, Water-Solubilizing Reverse Micelles, and Polar Organic Solvent Solubilizing Reverse Micelles. Langmuir, 2002, 18(21), 7872-7879. (88) Blanco, C.G.; Rodriguez, L.J.; Velazquez, M.M. Effect of the Solvent on the Water Properties of Water/Oil Microemulsions J. Colloid Interface Sci., 1999, 211, 380-386. (89) Megyesi, M.; Biczo´k, L. Effect of Ion Pairing on the Fluorescence of Berberine, a Natural Isoquinoline Alkaloid. Chem. Phys. Lett. 2007, 447, 247–251. (90) Dutt, G.B. Fluorescence Anisotropy of Ionic Probes in AOT Reverse Micelles: Influence of Water Droplet Size and Electrostatic Interactions on Probe Dynamics. J. Phys. Chem. B 2008, 112, 7220–7226. (91) Tan, H.S.; Piletic, I.R.; Fayer, M.D. Orientational Dynamics of Water Confined on a Nanometer Length Scale in Reverse Micelles. J. Chem. Phys. 2005,122, 174501-174509. (92) Pietrini, A.V.; Luisi, P.L. Circular Dichroic Properties and Average Dimensions of DNAContaining Reverse Micellar Aggregates. Biochim Biophys Acta 2002, 1562 57– 62. (93) Parodi, S.; Kendall, F.; Nicolin, C. A Clarification of the Complex Spectrum Observed with the Ultraviolet Circular Dichroism of Ethidium Bromide Bound to DNA. Nuc. Acids Res.,1975,2, 477-486. (94) Lyng, R.; Hard, T.; Norden, B. Induced CD of DNA Intercalators: Electric Dipole Allowed Transitions. Biopolymers, 1987, 26, 1327-1345. (95) Imre, V.E.; Luisi, P.L. Solubilization and Condensed Packing of Nucleic Acids in Reverse Micelles. Biochem Biophys Res Commun 1982, 107, 538–545. (96) Ainalema, M.L.; Nylander, T. DNA Condensation Using Cationic Dendrimers— Morphology and Supramolecular Structure of Formed Aggregates. Soft Matter, 2011, 7, 4577. (97) Conwell, C.C.; Vilfan, I.D.; Hud, N.V. Controlling the Size of Nanoscale Toroidal DNA Condensates with Static Curvature and Ionic Strength. Proc Natl Acad Sci USA 2003, 100, 9296–9301.


Page 26 of 37

Page 27 of 37

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


Scheme 1. Chemical Structure of Berberine Chloride, AOT and BHDC surfactants and ground state optimized structure of Berberine Chloride (the ground state geometry of BBCl was optimized using B3LYP function, using standard basis sets, 6-31G (d,p) for all atoms using Gausian-3)



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

Page 28 of 37

Table 1. Change in the Lifetime values of the Berberine Chloride in presence of different surfactants and CT- DNA.a rs

rt

ru

vs (ns)

vt (ns)

vu (ns)

W0 = 0.00

0.39

0.38

0.23

0.754

3.34

9.72

(ns) 3.80

W0 = 0.70

0.46

0.34

0.20

0.476

2.22

6.42

2.25

W0 = 1.40

0.49

0.30

0.21

0.370

1.59

4.72

1.65

W0 = 2.80

0.48

0.30

0.22

0.270

1.01

3.27

1.15

W0 = 6.2

0.48

0.30

0.22

0.210

0.720

2.25

0.81

W0 = 0.00

0.38

0. 38

0.24

0.40

2.11

5.03

2.16

W0 = 2.00

0.40

0.39

0.21

0.37

2.10

4.34

1.87

W0 =4.00

0.42

0.41

0.17

0.35

2.08

4.02

1.68

W0 =6.00

0.42

0.44

0.14

0.26

1.70

3.14

1.29

0.000mM

0.99

0.01

-

0.098

4.09

-

0.137

0.040 mM

0.72

0.20

0.08

0.144

1.38

5.50

0.819

0.150 mM

0.56

0.34

0.10

0.335

2.09

7.80

1.670

0.230 mM

0.55

0.35

0.10

0.376

2.20

8.18

1.790

0.340 mM 0.55 CT-DNA(0.5mM) in 0.43 BHDC RMs (W0=4.00) a experimental error ±5%

0.35 0.48

0.10 0.09

0.413 0.46

2.22 2.97

8.21 11.4

1.820 2.64

System

0.4 (M) AOT

0.4(M) BHDC

CTDNA

Table 2: Anisotropy Decay Data for BBCl in CT-DNAa. System BBCl in CT-DNA a

rs

0.22 experimental error ±5%

rt

vzs (ns)

vzt (ns)

v{ (ns)

|} (ns)

0.78

0.257

2.93

0.28

23.4


Page 29 of 37

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


Table 3: Fluorescence Anisotropy Decay Parameters of BBCl in AOT and BHDC RMs as a function of W0 a rs

System W0 = 0.00 W0 = 0.34 AOT RMs W0 = 0.68 W0 = 2.70 W0 = 6.20 W0 = 0.00 W0 = 2.00 BHDC RMs W0 = 4.00 W0 = 6.00 W0 = 10.00 CT-DNA (0.5mM) in BHDC RMs (W0 =4.00) a

rt

0.59 0.50 0.46 0.29 0.21 0.46 0.18 0.17 0.14 0.13 0.88

vzs (ns)

0.41 0.50 0.54 0.71 0.79 0.54 0.72 0.83 0.86 0.87 0.12

2.87 2.30 1.99 1.50 0.66 2.63 2.39 2.08 1.74 1.49 4.76

vzt (ns) 1.05 0.82 0.75 0.66 0.325 0.93 0.67 0.45 0.41 0.37 0.40

(ns) 2.12 1.56 1.32 0.90 0.40 1.71 0.91 0.73 0.60 0.52 4.23

experimental error ±5%

Table 4: Order Parameters, Cone Angles and Lateral and Wobbling Diffusion Coefficients for BBCl in AOT RMs obtained from Anisotropy Decay Parameters using Two-Step Model. W

(ns)

S

|

X 1012 (m2s-1)

{ X 10-8 (s-1)

0.00 0.34 0.68 2.77 6.20

2.30 2.53 2.77 4.58 9.10

0.76 0.70 0.68 0.53 0.45

340 380 410 500 550

35.4 75.38 121.0 462.0 1400

0.60 0.80 1.10 2.10 3.10

Table 5: Order Parameters, Cone Angles and Lateral and Wobbling Diffusion Coefficients for BBCl in BHDC RMs obtained from Anisotropy Decay Parameters using Two-Step Model. W

(ns)

S

|

X 1012 (m2s-1)

{ X 10-8 (s-1)

0.00 2.00 4.00 6.00 10.00

3.82 6.30 9.05 13.63 25.01

0.67 0.42 0.41 0.37 0.36

400 570 580 600 610

30.8 105.3 197.8 341.7 643.5

0.56 1.92 3.60 3.90 4.30



2.0

(b) Fluorescence Intensity (a.u)

Absorbance (a.u)

Fluorescence Intensity

800

(b) 1.6

0.000 mM CTDNA

1.2

0.8

0.340 mM CTDNA 0.4

600

700

400

600

200

500

0

0.0 0.1 0.2 0.3 Concnetration of CTDNA (mM)

400

0.340 mM CTDNA 300 200

0.000 mM CTDNA 100

0.0

0

250

300

350

400

450

500

500

550

Wavelength (nm)

550

600

650

700

Wavelength (nm)

Figure 1. Steady state absorption spectra (a) and fluorescence spectra (b) of Berberine chloride in presence of different concentration of CT DNA in phosphate buffer (pH = 7). (Inset in the figure (b) shows the change in the fluorescence intensity with addition of different concentration of CT-DNA)

40

(a)

50

Scatchard Plot from absorption spectra

(b)

Scatchard Plot from emission spectra

40 30

r/cf (mM-1)

r/cf (mM-1)

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

Page 30 of 37

20

10

30

20

10

0 0.22

0.24

0.26

0.28

0.30

0.32

0.34

0.36

0.24

0.28

0.32

0.36

r

r

Figure 2. Scatchard Plot for BBCl interaction with CT-DNA obtained from (a) absorption spectra and (b) emission spectra. Experimental data are shown by the black dot and the green line passes through the dots are drawn as visual aids and the red and blue line are plotted to determine n and Kb considering the possibility of existence of two independent non-cooperative binding sites.


0.40

Page 31 of 37

(a)

1.6 1.4 1.2

250

Ethidium Bromide -DNA

500

1.8

Fluorescence Intensity (a.u)

300

2.0

Barbarine Chloride-DNA F0/F

350


1.0 0.00

0.03

0.06

0.09

0.12

0.15

CNaI

200

0 (M) NaI

150 100

0.25 (M) NaI

(b)

0 (M) NaI

400

0.4 (M) NaI 300

200

100

50 0

0 500

550

600

650

500

700

550

600

650

700

Wavelength (nm)

Wavelength (nm)

400 350

3.0

(c)

2.5

300

I0/I


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


2.0 1.5

250

1.0

200 0.0

0.1

0.2

0.3

[NaCl]

150

0 mM NaCl

100 50 275 mM NaCl

0 500

550

600

650

700

750

800

Wavlength (nm)

Figure 3. Fluorescence Quenching of (a) BBCl-DNA complex (b) Ethidium Bromide-DNA complex in presence of NaI. Inset shows the Stern-Volmer plot in figure (a). (c) Effect of ionic strength on BBCl-DNA complex and inset show the Stern-Volmer plot against the concentration of NaCl.


750


Prompt CDNA =0 mM

Count (normalized)

1

CDNA =0.040 mM CDNA =0.113 mM 0.1

CDNA =0.340mM

0.01

0

10

20

30

40

Lifetime (ns)

Figure 4. Time Resolved Fluorescence Decays of BBCl in Phosphate Buffer (pH 7) in presence of different concentration of CT-DNA ( = )

0.25

(a)

20.7

ln (K nr)

20.1

0.012

(b)

0.20

20.4

0.015

Anisotropy

0.018

Quantum yield

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

Page 32 of 37

0.0028

0.0030

0.0032

0.0034

-1

1/T (k )

0.15

0.10

0.009

0.05

0.006 300

310

320

330

340

0.0

350

0.5

1.0

1.5

Concentration of DNA (mM)

Temp (k)

Figure 5. (a) Effect of temperature on the quantum yield of BBCl-DNA complex (inset shows the Arrhenius Plot of ln(z ) against 1/T) (b) Steady State Anisotropy of BBCl in presence of different concentration of CT-DNA.


Page 33 of 37

30

Elipticity (mdeg)

Ellipticity (mdeg)

30

20

10

15

0

-15 300

450

600

Wavelength (nm)

0

-10 200

250

300

350

Wavelength (nm) Figure 6. CD Spectra of 1.2 mM of CT-DNA in presence of 0 (black), 0.040 mM (green), 0.080 mM (blue), 0.160 mM (purple), 0.20 mM (red) Berberine Chloride (inset shows that very weak induce CD spectra is observed in presence of Berberine Chloride, shown by the circle)

0.9

(a)

AOT RMs

0.8

0.6

W0 = 7.00 0.3

(b)

BHDC RMs

W0 = 0

W0 = 0.00 Absorbance

Absorbance

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


0.6

0.4

W0 =11.8

0.2

0.0

0.0 320

360

400

440

480

300

Wavelength (nm)

350

400

450

Wavelength (nm)

Figure 7. Absorption Spectra of BBCl in (a) 0.4 (M) AOT RMs and (b) 0.4 (M) BHDC RMs in different W0 values.


500

Page 34 of 37

1200

(a)

AOT RMs


4000 3500

W0 = 0.00

3000 2500 2000

W0 = 7.00

1500 1000 500

(b)

BHDC RMs

1000

W0 = 0

800 600

W0 = 11.8

400 200

0

0

500

550

600

650

700

500

550

Wavelength (nm)

600

650

700

750

Wavelength (nm)

Figure 8: Steady State Fluorescence Measurement of BBCl in (a) 0.4 (M) AOT and (b) 0.4 (M) BHDC RMs in different W0 values. 1100

(a)

3500

AOT RMs

3000

540

2500 535 2000 530

1500

525

1000 500

Emission maxima (nm)

545

1

2

3

4

5

6

552

1050

551

1000

550

950

BHDC RMs

900

549

850

548 800 750

547

700

546

650

520 0

(b)

Emission maxima (nm)

550


4000

0

2

4

6

8

10

12

W0

7

W0 0.14

(c)

AOT RMs

BHDC RMs

0.12

Quantum Yield


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



0.10 0.08 0.06 0.04 0.02 0

2

4

6

8

10

12

W0

Figure 9. (a), (b) Change in the emission maxima and the fluorescence intensity of BBCl in different RMs and (c) Change in the quantum yield of BBCl in two reverse micelles in different W0 values.


Page 35 of 37

(a)

1

AOT RMs W0 = 0

0.1

0.01

W0 = 6.2 0

10

20

(b)

Prompt

30

Prompt W0 =0.00 W0 =2.00

BHDC RMs

Count (Normalized)

1

W0 =4.00 W0 =6.00

0.1

0.01

40

0

Time (ns)

10

20

30

40

Time (ns)

Figure 10. Time resolved Fluorescence Decays of BBCl in 0.4 (M) (a) AOT and (b) BHDC RMs in different W0 values.

0.4

0.4

(a) AOT RMs

w0 = 0.00

W0 = 0.00 W0 = 0.34

(b)

w0 = 0.34

0.3

BHDC RMs

0.3

w0 =2.77 w0 =6.20

0.2

r(t)

r (t)

Count (Normalized)

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


W0 = 0.68 W0 = 2.77 W0 = 6.20

0.2

0.1 0.1

0.0 0.0

0

2

4

6

0

2

4

6

8

Time (ns)

Time (ns)

Figure 11. Time Resolved Fluorescence Anisotropy of BBCl in 0.4 (M) (a) AOT and (b) BHDC RMs in different W0 values.



(c)

(d)

Figure 12. Fluorescence Lifetime Image of (a) BBCl/ BHDC ({ = . )/CT-DNA and (b) BBCl/AOT ({ = . )/CT-DNA. (Length of the scale bar is 50 µm and 25 µm in image (a) and (b) respectively). Both the images are taken in gray mode and FLIM mode. FESEM image of (c) BHDC RMs ({ = . )/CT-DNA (length of the scale bar is 300 nm) and (d) AOT RMs ({ = . )/CT-DNA (length of the scale bar is 5µm )

1

0.4

(a)

(b)

Buffer DNA

W0 = 4.00

W0 = 4.00

0.3

Prompt Buffer DNA

0.1

0.01

0.2

r (t)

count (Normalized)

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

Page 36 of 37

0.1

0.0

0

10

20

30

40

50

0

Time (ns)

2

4

6

Time (ns)

Figure 13. (a) Time Resolved Fluorescence Decays and (b) Time resolved Fluorescence Anisotropy of BBCl in BHDC RMs containing buffer and 0.5 mM CT-DNA at { = . . 36 ACS Paragon Plus Environment

8

Page 37 of 37

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


Table of Content (TOC) Only:


Unveiling the Mode of Interaction of Berberine ... - ACS Publications

Recommend Documents