Structure and the Anticancer Activity of Vitamin D Receptor Agonists.

Vitamin D is a group of seco-steroidal fat-soluble compounds. The two basic forms, vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol), do not have biological activity. They are converted in the body by a two-step enzymatic hydroxylation into biologically active forms, 1α,25-dihydroxyvitamin D2 [ercalcitriol, 1,25(OH)2D2] and 1α,25-dihydroxyvitamin D3 [calcitriol, 1,25(OH)2D3], which act as classical steroid hormones. 1,25(OH)2D3 exerts most of its physiological functions by binding to the nuclear vitamin D receptor (VDR), which is present in most body tissues to provide support to a broad range of physiological processes. Vitamin D-liganded VDR controls the expression of many genes. High levels of 1,25(OH)2D3 cause an increase in calcium in the blood, which can lead to harmful hypercalcemia. Several analogs of 1,25(OH)2D3 and 1,25(OH)2D2 have been designed and synthesized with the aim of developing compounds that have a specific therapeutic function, for example, with potent anticancer activity and a reduced toxic calcemic effect. Particular structural modifications to vitamin D analogs have led to increased anticancer activity and reduced calcemic action with the prospect of extending work to provide future innovative therapies.

Development of a film-forming oleogel with increased substantivity for the treatment of psoriasis.

The aim of this work was the development of a film-forming formulation (FFF) for the topical treatment of psoriasis that shows an increased substantivity compared to conventional semi-solid dosage forms. The developed formulation is an oleogel. It is based on a combination of castor oil and medium chain triglycerides, and the oil-soluble film former MP-30 (Croda GmbH, Nettetal, Germany), a polyamide that upon mixing with a polar oil entraps the oil und thus substantially increases the viscosity of the formulation up to a semisolid state. Betamethasone dipropionate (BDP) and calcipotriole (CA) were used as active pharmaceutical ingredients (APIs). Oleogels of different compositions were evaluated regarding substantivity, rheological properties, ex-vivo penetration into the skin and ex-vivo permeation through the skin. Marketed products were used as controls. It was found that the amount of betamethasone dipropionate penetrating and permeating into and through the skin from the film-forming formulation is at an intermediate value compared to the marketed products. The substantivity of the developed formulation is described by an amount of 57.7 % formulation that remains on the skin surface and is thus significantly higher compared to the marketed products. In the film forming formulation, the proportion of API penetrating the skin remains the same when the skin repetitively brought in contact with a piece of textile during the penetration experiment. In contrast with the in-market formulations tested, this proportion was reduced by up to 97 %. As a result, the developed formulations can lead to an increased patient compliance.

4-Hydroxy-1α,25-Dihydroxyvitamin D3: Synthesis and Structure-Function Study.

The active vitamin D metabolites, 25-hydroxyvitamin D3 (25D3) and 1,25-dihydroxyvitamin D3 (1,25D3), are produced by successive hydroxylation steps and play key roles in several cellular processes. However, alternative metabolic pathways exist, and among them, the 4-hydroxylation of 25D3 is a major one. This study aims to investigate the structure-activity relationships of 4-hydroxy derivatives of 1,25D3. Structural analysis indicates that 1,4α,25(OH)3D3 and 1,4ß,25(OH)3D3 maintain the anchoring hydrogen bonds of 1,25D3 and form additional interactions, stabilizing the active conformation of VDR. In addition, 1,4α,25D3 and 1,4ß,25D3 are as potent as 1,25D3 in regulating the expression of VDR target genes in rat intestinal epithelial cells and in the mouse kidney. Moreover, these two 4-hydroxy derivatives promote hypercalcemia in mice at a dose similar to that of the parent compound.

Design, synthesis, and biological activity of D-bishomo-1α,25-dihydroxyvitamin D3 analogs and their crystal structures with the vitamin D nuclear receptor.

The biologically active metabolite of vitamin D3 - calcitriol - is a hormone involved in the regulation of calcium-phosphate homeostasis, immunological processes and cell differentiation, being therefore essential for the proper functioning of the human body. This suggests many applications of this steroid in the treatment of diseases such as rickets, psoriasis and some cancers. Unfortunately, using therapeutic doses of calcitriol is associated with high concentrations of this compound which causes hypercalcemia. For this reason, new calcitriol analogs are constantly sought, devoid of calcemic effects but maintaining its beneficial properties. In this study, we present the synthesis of vitamin D derivatives characterized by an enlarged (seven-membered) ring D. Preparation of the designed vitamin D compounds required separate syntheses of crucial building blocks (C/D-rings fragments with side chain and rings A) which were combined by different methods, including Wittig-Horner reaction and Suzuki coupling. Biological activities of the target vitamin D analogs were assessed both in vitro and in vivo, demonstrating their significant potency compared to the natural hormone. Furthermore, the successful crystallization of these compounds with the vitamin D receptor (VDR) enabled us to investigate additional molecular interactions with this protein.

Structural analysis and biological activities of C25-amino and C25-nitro vitamin D analogs.

Intense synthetic efforts have been directed towards the development of noncalcemic analogs of 1,25-dihydroxyvitamin D3. We describe here the structural analysis and biological evaluation of two derivatives of 1,25-dihydroxyvitamin D3 with modifications limited to the replacement of the 25-hydroxyl group by a 25-amino or 25-nitro groups. Both compounds are agonists of the vitamin D receptor. They mediate biological effects similar to 1,25-dihydroxyvitamin D3, the 25-amino derivative being the most potent one while being less calcemic than 1,25-dihydroxyvitamin D3. The in vivo properties of the compounds make them of potential therapeutic value.

Novel superagonist analogs of 2-methylene calcitriol: Design, molecular docking, synthesis and biological evaluation.

A new series of highly biologically active (20S,22R)-1α,25-dihydroxy-22-methyl-2-methylene-vitamin D3 analogs, possessing different side chains, have been efficiently prepared as potential agents for medical therapy. Design of these synthetic targets was based on the analysis of the literature data and molecular docking experiments. The synthetic strategy involved Sonogashira coupling of the known A-ring dienyne with the C,D-ring enol triflates, obtained from the corresponding Grundmann ketones. All synthesized vitamin D compounds were characterized by high in vitro potency and, moreover, they proved to be very calcemic in vivo exerting high activity on bone with particularly elevated intestinal calcium transport.

Biological evaluation and synthesis of calcitroic acid.

We describe the synthesis and broad profiling of calcitroic acid (CTA) as vitamin D receptor (VDR) ligand. The x-ray co-crystal structure of the Danio Rerio VDR ligand binding domain in complex with CTA and peptide MED1 confirmed an agonistic conformation of the receptor. CTA adopted a similar conformation as 1,25(OH)2D3 in the binding pocket. A hydrogen bond with His333 and a water molecule were observed in the binding pocket, which was accommodated due to the shorter CTA side chain. In contrast, 1,25(OH)2D3 interacted with His423 and His333 due to its longer side chain. In vitro, the EC50 values of CTA and CTA-ME for VDR-mediated transcription were 2.89 µM and 0.66 µM, respectively, confirming both compounds as VDR agonists. CTA was further evaluated for interaction with fourteen nuclear receptors demonstrating selective activation of VDR. VDR mediated gene regulation by CTA in intestinal cells was observed for the VDR target gene CYP24A1. CTA at 10 µM upregulated CYP24A1 with similar efficacy as 1,25(OH)2D3 at 20 nM and 100-fold stronger compared to lithocholic acid at 10 µM. CTA reduced the transcription of iNOS and IL-1ß in interferon γ and lipopolysaccharide stimulated mouse macrophages resulting in a reduction of nitric oxide production and secretion of IL-1ß. These observed anti-inflammatory properties of 20 µM CTA were similar to 20 nM 1,25(OH)2D3.

Efficiency of Gemini surfactant containing semi-rigid spacer as microbial corrosion inhibitor for carbon steel in simulated seawater.

SRB is one of the main bacteria causing marine microbial corrosion. In order to reduce the loss of microbial corrosion, a Gemini surfactant (12-B-12) containing semi-rigid spacer was used to investigate the anti-bacterial and anti-corrosion performances of carbon steel in simulated seawater by weight-loss test, electrochemical method and surface morphology analysis. The results showed that the inhibition efficiency of 0.01 mM 12-B-12 was as high as 98.3% after 30 days of incubation in simulated seawater with SRB, and the planktonic and sessile SRB on the carbon steel surface can be reduced to undetectable level. Quantum chemical calculation and molecular dynamics simulation were used to study the structure-activity relationship.

Photoresponsive transformation from spherical to nanotubular assemblies: anticancer drug delivery using macrocyclic cationic gemini amphiphiles.

We developed NIR-light-responsive macrocyclic cationic gemini amphiphiles, one of which displayed various favorable properties of lipids. The NIR-light-mediated cleavage of the strained dioxacycloundecine ring led to the conversion of the spherical to a nanotubular self-assembly in the aqueous medium. This photo-mediated transformation from the spherical to nanotubular self-assembly resulted in the release of encapsulated hydrophobic anticancer drug molecule doxorubicin (Dox) in a controlled manner. The potent cationic gemini amphiphile also displayed lower cytotoxicity and efficient NIR-light-mediated Dox release efficacy to cancerous cells.

Proposal of novel inhibitors for vitamin-D receptor: Molecular docking, molecular mechanics and ab initio molecular orbital simulations.

The specific binding of active vitamin-D to the vitamin-D receptor (VDR) is closely related to the onset of immunological diseases. To inhibit the binding, various compounds have been developed as potent inhibitors against VDR. Among them, a compound NS-54c, which was developed based on the first VDR antagonist TEI-9647 (25-dehydro-1α-hydroxyvitamin D3-26,23-lactone), was revealed to posse almost 1000-fold improved antagonistic activity over the original TEI-9647. However, the reason for this significant improvement has not been elucidated. In the present study, we investigated the specific interactions between VDR and these inhibitors, using molecular simulations based on molecular docking, molecular mechanics and ab initio fragment molecular orbital calculations. Based on the results simulated, we furthermore proposed novel inhibitors and investigated their binding properties to VDR. The results elucidate that the replacement of propyl group at the 24th site of NS-54c by a phenethyl group can enhance the binding affinity of the inhibitor to VDR. This finding provides useful information for developing novel potent inhibitors against VDR.

Gemini Curcumin Suppresses Proliferation of Ovarian Cancer OVCAR-3 Cells via Induction of Apoptosis.

BACKGROUND: Ovarian cancer has the highest mortality rate among gynecological malignancies. Despite recent advances in treatment, most patients still suffer from poor prognosis. Curcumin has shown highly cytotoxic effects against different types of cancer. However, its poor bioavailability restricts its clinical application. Gemini Curcumin (Gemini-Cur) has been developed to overcome this limitation. OBJECTIVE: Here, we aimed to unravel the inhibitory effect of Gemini-Cur in ovarian cancer. METHODS: OVCAR-3 cells were treated with free curcumin and Gemni-Cur in a time- and dose-dependent manner. Then, the anticancer activity was investigated by uptake kinetics, cellular viability and apoptotic assays. Furthermore, we evaluated the BAX/Bcl-2 expression ratio by real-time PCR and western blotting. RESULTS: Our data showed that gemini surfactant nanoparticles enhance the cellular uptake of curcumin compared to free curcumin (p<0.01). Regarding the growth inhibitory effect of nano-curcumin, the results demonstrated that Gemini-Cur suppresses the proliferation of OVCAR-3 cells through induction of apoptosis (p<0.001). CONCLUSION: The results illustrate that Gemini-Cur nanoparticles have a great potential for developing novel therapeutics against ovarian cancer.

Structure-Activity Relationship Study of an Alkynylphosphonate and Vynilphosphonate Analogues of Calcitriol.

BACKGROUND: 1α,25-dihydroxy vitamin D3 (calcitriol) shows potent growth-inhibitory properties on different cancer cell lines, but its hypercalcemic effects have severely hampered its therapeutic application. Therefore, it is important to develop synthetic calcitriol analogues that retain or even increase its antitumoral effects and lack hypercalcemic activity. Based on previous evidence of the potent antitumor effects of the synthetic alkynylphosphonate EM1 analogue, we have now synthesized a derivative called SG. OBJECTIVE: The aim of the present work is to evaluate the calcemic activity and the antitumor effect of SG, comparing these effects with those exerted by calcitriol and with those previously published for EM1. In addition, we propose to analyze by in silico studies, the chemical structure-biological function relationship of these molecules. METHODS: We performed the synthesis of vinylphosphonate SG analogue; in vitro assays on different cancer cell lines; in vivo assays on mice; and in silico assays applying computational molecular modeling. RESULTS: The SG compound lacks hypercalcemic activity, similar to the parent compound EM1. However, the antitumor activity was blunted, as no antiproliferative or anti-migratory effects were observed. By in silico assays, we demonstrated that SG analogue has a lower affinity for the VDRligand- binding domain than the EM1 compound due to lack of interaction with the important residues His305 and His397. CONCLUSION: These results demonstrate that the chemical modification in the lateral side chain of the SG analogue affects the antitumoral activity observed previously for EM1 but does not affect the calcemic activity. These results contribute to the rational design and synthesis of novel calcitriol analogues.

Immunomodulatory Dual-Sized Microparticle System Conditions Human Antigen Presenting Cells Into a Tolerogenic Phenotype In Vitro and Inhibits Type 1 Diabetes-Specific Autoreactive T Cell Responses.

Current monotherapeutic agents fail to restore tolerance to self-antigens in autoimmune individuals without systemic immunosuppression. We hypothesized that a combinatorial drug formulation delivered by a poly-lactic-co-glycolic acid (PLGA) dual-sized microparticle (dMP) system would facilitate tunable drug delivery to elicit immune tolerance. Specifically, we utilized 30 µm MPs to provide local sustained release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor ß1 (TGF-ß1) along with 1 µm MPs to facilitate phagocytic uptake of encapsulated antigen and 1α,25(OH)2 Vitamin D3 (VD3) followed by tolerogenic antigen presentation. We previously demonstrated the dMP system ameliorated type 1 diabetes (T1D) and experimental autoimmune encephalomyelitis (EAE) in murine models. Here, we investigated the system's capacity to impact human cell activity in vitro to advance clinical translation. dMP treatment directly reduced T cell proliferation and inflammatory cytokine production. dMP delivery to monocytes and monocyte-derived dendritic cells (DCs) increased their expression of surface and intracellular anti-inflammatory mediators. In co-culture, dMP-treated DCs (dMP-DCs) reduced allogeneic T cell receptor (TCR) signaling and proliferation, while increasing PD-1 expression, IL-10 production, and regulatory T cell (Treg) frequency. To model antigen-specific activation and downstream function, we co-cultured TCR-engineered autoreactive T cell "avatars," with dMP-DCs or control DCs followed by ß-cell line (ßlox5) target cells. For G6PC2-specific CD8+ avatars (clone 32), dMP-DC exposure reduced Granzyme B and dampened cytotoxicity. GAD65-reactive CD4+ avatars (clone 4.13) exhibited an anergic/exhausted phenotype with dMP-DC presence. Collectively, these data suggest this dMP formulation conditions human antigen presenting cells toward a tolerogenic phenotype, inducing regulatory and suppressive T cell responses.

Protection from Ultraviolet Damage and Photocarcinogenesis by Vitamin D Compounds.

Exposure of skin cells to UV radiation results in DNA damage, which if inadequately repaired, may cause mutations. UV-induced DNA damage and reactive oxygen and nitrogen species also cause local and systemic suppression of the adaptive immune system. Together, these changes underpin the development of skin tumours. The hormone derived from vitamin D, calcitriol (1,25-dihydroxyvitamin D3) and other related compounds, working via the vitamin D receptor and at least in part through endoplasmic reticulum protein 57 (ERp57), reduce cyclobutane pyrimidine dimers and oxidative DNA damage in keratinocytes and other skin cell types after UV. Calcitriol and related compounds enhance DNA repair in keratinocytes, in part through decreased reactive oxygen species, increased p53 expression and/or activation, increased repair proteins and increased energy availability in the cell when calcitriol is present after UV exposure. There is mitochondrial damage in keratinocytes after UV. In the presence of calcitriol, but not vehicle, glycolysis is increased after UV, along with increased energy-conserving autophagy and changes consistent with enhanced mitophagy. Reduced DNA damage and reduced ROS/RNS should help reduce UV-induced immune suppression. Reduced UV immune suppression is observed after topical treatment with calcitriol and related compounds in hairless mice. These protective effects of calcitriol and related compounds presumably contribute to the observed reduction in skin tumour formation in mice after chronic exposure to UV followed by topical post-irradiation treatment with calcitriol and some, though not all, related compounds.

Differential Impact of Calcitriol and Its Analogs on Tumor Stroma in Young and Aged Ovariectomized Mice Bearing 4T1 Mammary Gland Cancer.

(1) Background: Vitamin D compounds (VDC) are extensively studied in the field of anticancer properties, including breast cancer. Previously, we showed that calcitriol and its analogs (PRI-2191 and PRI-2205) stimulate metastasis in 4T1 murine mammary gland cancer models in young mice, whereas the reverse effect was observed in aged ovariectomized (OVX) mice; (2) Methods: We determined the phenotype of monocytes/macrophages using FACS and examined the expression of selected genes and proteins by Real-Time PCR and ELISA; (3) Results: Activities of VDC are accompanied by an increase in the percentage of Ly6Clow anti-inflammatory monocytes in the spleen of young and a decrease in aged OVX mice. Treatment of young mice with VDC resulted in an increase of CCL2 plasma and tumor concentration and Arg1 in tumor. In later stage of tumor progression the expression of genes related to metastasis in lung tissue was decreased or increased, in old OVX or young mice, respectively; (4) Conclusions: Pro- or anti-metastatic effects of calcitriol and its analogs in young or aged OVX mice, respectively, can be attributed to the differences in the effects of VDC on the tumor microenvironment, as a consequence of differences in the immunity status of young and aged mice.

[12]aneN3-Based Gemini-Type Amphiphiles with Two-Photon Absorption Properties for Enhanced Nonviral Gene Delivery and Bioimaging.

Although a plethora of nonviral gene vectors have been developed for potential gene therapy, imageable gemini surfactants with stimuli-responsiveness and high transfection efficiency are still scarce for gene delivery. Herein, three gemini amphiphiles (DEDPP-4/8/12) consisting of an aggregation-induced emission (AIE) central fluorophore: 5,6-diphenylpyrazine-2,3-diester (DEDPP), decorated with triazole-[12]aneN3 as the hydrophilic moiety and alkyl chains of various lengths as the hydrophobic moiety, were designed and synthesized for trackable gene delivery via optical imaging. All three amphiphiles exhibited ultralow critical micelle concentrations (CMCs) (up to 3.40 × 10-6 M), prominent two-photon absorption properties, and solvatochromic fluorescence. Gel electrophoresis assays demonstrated that the migration of plasmid DNA was completely retarded after condensation with these gemini amphiphiles at low concentrations (up to 10 µM). In addition, the ester bond in these amphiphiles may facilitate vector degradation and DNA release, in response to esterase and the acidic environment inside cells. Upon self-assembly with DOPE to form liposomes, DEDPP-8/DOPE achieved the best transfection efficiency in four cell lines, and the transfection efficiency of DEDPP-8/DOPE in HeLa cell lines was 23.5-fold higher than that of Lipo2000, which is unusually high for small organic molecule-based nonviral vectors. Furthermore, excellent transfection efficiency of DEDPP-8/DOPE was obtained in the presence of serum, and the red fluorescence protein (RFP) gene was successfully transfected in zebrafish embryos. Both one- and two-photon fluorescence imaging clearly demonstrated the delivery process of plasmid DNA. This study demonstrated that gemini-type amphiphiles composed of a two-photon fluorophore core conjugated with triazole-[12]aneN3 via an ester bond afforded an unprecedentedly high transfection efficiency with excellent biocompatibility, which may provide new insights for the design and development of multifunctional nonviral gene vectors for imageable gene delivery.

Design, synthesis and biological evaluation of novel 2-alkylidene 19-norcalcitriol analogs.

Continuing our studies aimed at A-ring modified vitamin D compounds, we designed novel 19-norcalcitriol derivatives bearing at C-2 pegylated chains of different lengths. The terminal fragments of these substituents contain hydroxyls or moieties possessing nitrogen and/or sulfur atoms capable of transition metal ions complexation. Also, two conjugate-type platinum(II) complexes of 19-norcalcitriol were obtained in which l-methionine served as chelating moiety. The convergent synthesis of the target 19-norcalcitriol analogs involved several steps with the crucial one being condensation of A-ring phosphine oxide and the known Grundmann ketone by Wittig-Horner reaction. Further elaboration of the 2-alkylidene substituent provided all final compounds which were then tested to determine their affinity for the vitamin D receptor and cytotoxic activity.

A sensitive "Switch-on" phosphorescent probe for ferrous iron quantification in drug and In vitro imaging of living cells.

Iron plays an important role in various physiological processes. However, the detailed biological functions of iron have not been sufficiently explored because of a lack of effective methods to monitoring iron, especially the labile ferrous ion (Fe2+). In the current study, a novel turn-on phosphorescent probe for Fe2+ quantification and visualization has been proposed based on the hybrid nanocomposite of manganese dioxide and gemini iridium complex (MnO2-GM-Ir). The surfactant-like GM-Ir with positive charges was beneficial to combine with the negatively charged manganese dioxide (MnO2) nanosheets, and thus endowing the MnO2-GM-Ir nanocomposite excellent dispersion ability in the water as well as efficiently avoiding the interference to the detection caused by the agglomeration of nanocomposite. Phosphorescence of GM-Ir was effectively quenched by MnO2 nanosheets through fluorescence resonance energy transfer (FRET) and the inner filter effect (IFE), while the phosphorescence could be significantly recovered in the presence of Fe2+via a selective Fe2+-mediated reduction of MnO2 nanosheets, indicating a highly-specific selectivity towards Fe2+ with a low detection limit (80 nM). The drug test assay and in vitro imaging studies further proved that the MnO2-GM-Ir nanocomposite could be employed as a promising probe for the quantitative detection of exogenous Fe2+ in drug and in vitro imaging of living cells.

Antiproliferative activity of side-chain truncated vitamin D analogs (PRI-1203 and PRI-1204) against human malignant melanoma cell lines.

Vitamin D compounds are versatile molecules widely considered as promising agents in cancer prevention and treatment, including melanoma. Previously we investigated series of double point modified vitamin D2 analogs as well as non-calcemic 20S-hydroxyvitamin D3 and 21-hydroxypregnacalciferol as to their anti-melanoma activity. Surprisingly, short side-chain vitamin D analogs were found to be biologically active compounds. Thus, here we tested novel derivatives of pregnacalciferol with an additional hydroxyl at the end of the truncated side chain, PRI-1203 and PRI-1204, as to their potency against human melanoma A375 and RPMI7951 cell lines. Tested compounds are geometric isomers, with 19-methylene positioned in PRI-1203 like in a calcitriol molecule, but reversed in the PRI-1204 analog to the (5E,7E) geometry (5,6-trans). We noticed a decrease in cells viability exerted by PRI-1203. The antiproliferative effect of PRI-1204 was very low, emphasizing the importance of the natural 19-methylene geometry in the PRI-1203. PRI-1203 was also effective in inhibition of A375 melanoma cells migration. PRI-1203, but not PRI-1204, increased the percentage of A375 and RPMI7951 melanoma cells in the G0/G1 phase of cell cycle, possibly in a p21 and p27 independent manner. Both, analogs have very low effect on the level of CYP24A1 mRNA, in comparison to active form of vitamin D - 1.25(OH)2D3. In addition, both tested compounds failed to elicit VDR translocation to the nucleus. Thus, it could be postulated that side chain shortening strongly affects binding of analogs to VDR and activation of genomic responses, however do not impair their antiproliferative activities.

Interactions with ctDNA of novel sugar-based gemini cationic surfactants.

The interactions between calf thymus DNA, ctDNA, and a series of sugar-based gemini cationic surfactants with different hydrophobic chains were investigated. The surface properties of the cationic gemini surfactants were firstly examined, and then their interactions with DNA and induced condensation of DNA were studied by UV-vis, ethidium bromide exclusion assay, circular dichroism, dynamic light scattering, zeta potential and atomic force microscopy. With the increase of hydrophobic chains of the surfactants, critical micelle concentrations decreased significantly, and the interactions with DNA were remarkably strengthened, with the binding constant up to 1.95 × 107 L·mol-1 according to fluorescence quenching experiments by ethidium bromide exclusion. The gemini surfactant with hexadecyl hydrocarbon chain, 1c, exhibited the highest compaction capacity for DNA, accompanied with conformation changes, as confirmed by CD and DLS measurements. The DNA molecules could be compacted to about 140 nm in hydrodynamic diameter at 0.2 mM of 1c, and the overall shifts of the positive band and significant increase of negative molar ellipticity indicated the formation of a supramolecualr chiral order of ѱ phase in which DNA were supposed to be tightly packed.