D. Agudelo

Intercalation of antitumor drug doxorubicin and its analogue by DNA duplex: Structural features and biological implications

The intercalation of antitumor drug doxorubicin (DOX) and its analogue N-(trifluoroacetyl) doxorubicin (FDOX) with DNA duplex was investigated, using FTIR, CD, fluorescence spectroscopic methods and molecular modeling. Both DOX and FDOX were intercalated into DNA duplex with the free binding energy of -4.99 kcal for DOX-DNA and -4.92 kcal for FDOX-DNA adducts and the presence of H-bonding network between doxorubicin NH2 group and cytosine-19. Spectroscopic results showed FDOX forms more stable complexes than DOX with KDOX-DNA=2.5(± 0.5)× 10(4)M(-1) and KFDOX-DNA=3.4(± 0.7)× 10(4)M(-1). The number of drug molecules bound per DNA (n) was 1.2 for DOX and 0.6 for FDOX. Major alterations of DNA structure were observed by DOX intercalation with a partial B to A-DNA transition, while no DNA conformational changes occurred upon FDOX interaction. This study further confirms the importance of unmodified daunosamine amino group for optimal interactions with DNA. The results of in vitro MTT assay carried out on SKC01 colon carcinoma corroborate the observed DNA interactions. Such DNA structural changes can be related to doxorubicin antitumor activity, which prevents DNA duplication.

Effect of polymer molecular weight on chitosan-protein interaction.

We present a comprehensive study of the interactions between chitosan nanoparticles (15, 100 and 200 kDa with the same degree of deacetylation 90%) and two model proteins, i.e., bovine (BSA) and human serum albumins (HSA), with the aim of correlating chitosan molecular weight (Mw) and the binding affinity of these naturally occurring polymers to protein. The effect of chitosan on the protein secondary structure and the influence of protein complexation on the shape of chitosan nanoparticles are discussed. A combination of multiple spectroscopic methods, transmission electron microscopy (TEM) and thermodynamic analysis were used to assess the polymer-protein complex formation. Results revealed that the three chitosan nanoparticles interact with BSA to form chitosan-BSA complexes, mainly through hydrophobic contacts with the affinity order: 200>100>15 kDa. However, HSA-chitosan complexation is mainly via electrostatic interactions with the stability order: 100>200>15 kDa. Furthermore, the association between polymer and protein causes a partial protein conformational change by a major reduction of α-helix from 63% (free BSA) to 57% (chitosan-BSA) and 57% (free HSA) to 51% (chitosan-HSA). Finally, TEM micrographs clearly revealed that the binding of serum albumins with chitosan nanoparticles induces a significant change in protein morphology and the shape of the polymer.

Encapsulation of antitumor drug Doxorubicin and its analogue by chitosan nanoparticles.

Biodegradable chitosan of different sizes were used to encapsulate antitumor drug doxorubicin (Dox) and its N-(trifluoroacetyl) doxorubicin (FDox) analogue. The complexation of Dox and FDox with chitosan 15, 100, and 200 KD was investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods, and molecular modeling. The structural analysis showed that Dox and FDox bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K(Dox-ch-15) = 8.4 (±0.6) × 10(3) M(-1), K(Dox-ch-100) = 2.2 (±0.3) × 10(5) M(-1), K(Dox-ch-200) = 3.7 (±0.5) × 10(4) M(-1), K(FDox-ch-15) = 5.5 (±0.5) × 10(3) M(-1), K(FDox-ch-100) = 6.8 (±0.6) × 10(4) M(-1), and K(FDox-ch-200) = 2.9 (±0.5) × 10(4) M(-1), with the number of drug molecules bound per chitosan (n) ranging from 1.2 to 0.5. The order of binding is ch-100 > 200 > 15 KD, with stronger complexes formed with Dox than FDox. The molecular modeling showed the participation of polymer charged NH(2) residues with drug OH and NH(2) groups in the drug-polymer adducts. The presence of the hydrogen-bonding system in FDox-chitosan adducts stabilizes the drug-polymer complexation, with the free binding energy of -3.89 kcal/mol for Dox and -3.76 kcal/mol for FDox complexes. The results show chitosan 100 KD is a more suitable carrier for Dox and FDox delivery.

Microscopic and thermodynamic analysis of PEG–β-lactoglobulin interaction

We report the binding of milk β-lactoglobulin (β-LG) with PEG-3000, PEG-6000 and methoxypoly(ethylene glycol) anthracene (mPEG-anthracene) in aqueous solution at pH 7.4, using multiple spectroscopic methods, thermodynamic analysis, transmission electron microscopy (TEM) and molecular modeling. Thermodynamic and spectroscopic analysis showed that polymers bind β-LG via van der Waals interactions, hydrogen bonding and hydrophobic interactions, with overall binding constants KPEG-3000–β-LG = 9.2 (±0.9) × 103 M−1, KPEG-6000–β-LG = 9.7 (±0.7) × 103 M−1 and KmPEG-anthracene–β-LG = 5.5 (±0.5) × 104 M−1. The binding affinity was mPEG-anthracene > PEG-6000 > PEG-3000. Transmission electron microscopy analysis showed significant changes in protein morphology as polymer–protein complexation occurred, with a major increase in the diameter of the protein aggregate. Modeling showed several hydrogen bonding systems between PEG and the different amino acid stabilized polymer–β-LG complexes. The free binding energy indicated that the interaction process is spontaneous at room temperature. Furthermore, mPEG-anthracene is a stronger protein binder than PEG-3000 and PEG-6000, due to its major hydrophobic characteristics.

The role of polymer size and hydrophobic end-group in PEG-protein interaction.

We investigated the interaction between polyethylene (glycol) (PEG) and human (HSA) and bovine serum albumin (BSA) in aqueous solution, using multiple spectroscopic methods and molecular modeling. The two important polymer characteristics, size and PEG hydrophobic end-group are studied in order to determine the effect of each one on PEG-protein interaction. The bindings of PEG and mPEG-anthracene with serum albumins occur via hydrophobic and H-bonding contacts with the binding affinity PEG-6000>mPEG-anthracene>PEG-3000 for BSA and EG-6000>PEG-3000>mPEG-anthracene for HSA. Modeling showed different protein binding sites are involved in PEG-BSA and PEG-HSA complexes. Several H-bonding systems between PEG and different amino acids are stabilizing polymer-protein complexes. The free binding energies of -6.48 (PEG-BSA) and -6.36 kcal/mol (PEG-HSA) showed that the interaction process is spontaneous at room temperature. Minor alterations of protein alpha-helix and beta-sheet structures were observed upon PEG complexation.

Encapsulation of Milk β-Lactoglobulin by Chitosan Nanoparticles

Naturally occurring polymers, such as chitosan, have been extensively studied as carriers for therapeutic protein and gene delivery systems. β-Lactoglobulin (β-LG) is a member of the lipocalin superfamily of transporters for small hydrophobic molecules. We examine the binding of milk β-lactoglobulin with chitosan of different sizes such as chitosan 15, 100, and 200 KD in aqueous solution at pH 5-6, using FTIR, CD, and fluorescence spectroscopic methods. Structural analysis showed that chitosan binds β-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K(β-LG-ch-15) = 4.1 (±0.4) × 10(2) M(-1), K(β-LG-ch-100) = 7.2 (±0.6) × 10(4) M(-1), and K(β-LG-ch-200) = 3.9 (±0.5) × 10(3) M(-1) with the number of bound protein per chitosan (n) 0.9 for ch-15, 0.6 for ch-100, and 1.6 for ch-200. Chitosan 100 KD forms stronger complexes with β-LG than chitosans 200 and 15 KD. Polymer binding did not alter protein conformation inducing structural stabilization. Chitosan 100 is a stronger protein transporter than chitosan 15 and 200 KD.

tRNA Binding to Antitumor Drug Doxorubicin and Its Analogue

The binding sites of antitumor drug doxorubicin (DOX) and its analogue N-(trifluoroacetyl) doxorubicin (FDOX) with tRNA were located, using FTIR, CD, fluorescence spectroscopic methods and molecular modeling. Different binding sites are involved in drug-tRNA adducts with DOX located in the vicinity of A-29, A-31, A-38, C-25, C-27, C-28, G-30 and U-41, while FDOX bindings involved A-23, A-44, C-25, C-27, G-24, G-42, G-53, G-45 and U-41 with similar free binding energy (-4.44 for DOX and -4.41 kcal/mol for FDOX adducts). Spectroscopic results showed that both hydrophilic and hydrophobic contacts are involved in drug-tRNA complexation and FDOX forms more stable complexes than DOX with K DOX-tRNA = 4.7 (±0.5)×104 M−1 and K FDOX-tRNA = 6.3 (±0.7)×104 M−1. The number of drug molecules bound per tRNA (n) was 0.6 for DOX and 0.4 for FDOX. No major alterations of tRNA structure were observed and tRNA remained in A-family conformation, while biopolymer aggregation and particle formation occurred at high drug concentrations.

An overview on the delivery of antitumor drug doxorubicin by carrier proteins

Serum proteins play an increasing role as drug carriers in the clinical settings. In this review, we have compared the binding modalities of anticancer drug doxorubicin (DOX) to three model carrier proteins, human serum albumin (HSA), bovine serum albumin (BSA) and milk beta-lactoglobulin (β-LG) in order to determine the potential application of these model proteins in DOX delivery. Molecular modeling studies showed stronger binding of DOX with HSA than BSA and β-LG with the free binding energies of -10.75 (DOX-HSA), -9.31 (DOX-BSA) and -8.12kcal/mol (DOX-β-LG). Extensive H-boding network stabilizes DOX-protein conjugation and played a major role in drug-protein complex formation. DOX complexation induced major alterations of HSA and BSA conformations, while did not alter β-LG secondary structure. The literature review shows that these proteins can potentially be used for delivery of DOX in vitro and in vivo.

Transporting Antitumor Drug Tamoxifen and Its Metabolites, 4-Hydroxytamoxifen and Endoxifen by Chitosan Nanoparticles

Synthetic and natural polymers are often used as drug delivery systems in vitro and in vivo. Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K tam-ch-15  = 8.7 (±0.5)×103 M−1, K tam-ch-100  = 5.9 (±0.4)×105 M−1, K tam-ch-200  = 2.4 (±0.4)×105 M−1 and K hydroxytam-ch-15  = 2.6(±0.3)×104 M−1, K hydroxytam – ch-100  = 5.2 (±0.7)×106 M−1 and K hydroxytam-ch-200  = 5.1 (±0.5)×105 M−1, K endox-ch-15  = 4.1 (±0.4)×103 M−1, K endox-ch-100  = 1.2 (±0.3)×106 M−1 and K endox-ch-200  = 4.7 (±0.5)×105 M−1 with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH2 residues with drug OH and NH2 groups in the drug-polymer adducts. The free binding energies of −3.46 kcal/mol for tamoxifen, −3.54 kcal/mol for 4-hydroxytamoxifen and −3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD.

Microscopic and spectroscopic analysis of chitosan–DNA conjugates

Conjugations of DNA with chitosans 15 kD (ch-15), 100 kD (ch-100) and 200 kD (ch-200) were investigated in aqueous solution at pH 5.5-6.5. Multiple spectroscopic methods and atomic force microscopy (AFM) were used to locate the chitosan binding sites and the effect of polymer conjugation on DNA compaction and particle formation. Structural analysis showed that chitosan-DNA conjugation is mainly via electrostatic interactions through polymer cationic charged NH2 and negatively charged backbone phosphate groups. As polymer size increases major DNA compaction and particle formation occurs. At high chitosan concentration major DNA structural changes observed indicating a partial B to A-DNA conformational transition.