Graph represents the XRD pattern of synthesized MNPs shows the formation of Fe₃O₄ based on the comparison of their XRD patterns with the standard pattern of Fe₃O₄ (04-013-9808).

<p>The diffraction peaks are quite identical to characteristic peaks of the Fe₃O₄ crystal with the cubic spinal structure.</p

HR-TEM image of the prepared MNPs capped with ascorbic acid shows that particles have spherical shape with average size of 20.0±2.0 nm.

<p>HR-TEM image of the prepared MNPs capped with ascorbic acid shows that particles have spherical shape with average size of 20.0±2.0 nm.</p

Bars represent distribution of iron in skeletal muscles and ESC tumor tissue of Ehrlich bearing mice groups.

<p>Asterisks indicate a statistically significant difference compared with other groups.</p

Images of plates containing DH5-α bacteria transformed with A) Gel.NPs only, B) recombinant <i>NS2</i> gene only, C) labeled conjugate recombinant <i>NS2</i> Gel.NPs with three ratios (w/w) 1:50, 2:50 and 1:100.

<p>Images of plates containing DH5-α bacteria transformed with A) Gel.NPs only, B) recombinant <i>NS2</i> gene only, C) labeled conjugate recombinant <i>NS2</i> Gel.NPs with three ratios (w/w) 1:50, 2:50 and 1:100.</p

The image of <i>NS2</i>/Gel.NPs transformed DH5-α bacteria by Confocal Laser Scanning Microscope showed high number of colonies in yellow-orange color after laser excitation at 633 nm and 514 nm simultaneously.

<p>The image of Gel.NPs transformed into DH5-α bacteria showed no appearance of any emission colors due to absence of plasmid carrying ampicillin resistant gene, where bacteria died when grown in presence of ampicillin (Fig 5A). <i>NS2</i> transformed DH5-α bacteria showed lower number of bacterial colonies in green color after laser excitation at 514nm (Fig 5B). The yellow-orange color due to the cross over between the red labeled Gel.NPs and green labeled <i>NS2</i> gene could be detected in (Fig 5C) which indicates that presence of gelatin nanoparticles enhance transformation efficiency than using naked <i>NS2</i> in (Fig 5B).</p

Agarose gel electrophoresis for miniprep samples of the recombinant PQE 30/<i>NS2</i> plasmid/Gel.NPs double digested (<i>SphI</i>/<i>HindIII</i>) showing two bands; band for <i>NS2</i> insert (650 bp) and band for pQE-30 vector (3.4 kb) in Lane 1 and 2.

<p>Lane M: 1 kb DNA marker.</p

Gelatin nanoparticles enhance delivery of hepatitis C virus recombinant <i>NS2</i> gene

<div><p>Background</p><p>Development of an effective non-viral vaccine against hepatitis C virus infection is of a great importance. Gelatin nanoparticles (Gel.NPs) have an attention and promising approach as a viable carrier for delivery of vaccine, gene, drug and other biomolecules in the body.</p><p>Aim of work</p><p>The present study aimed to develop stable Gel.NPs conjugated with nonstructural protein 2 (<i>NS2</i>) gene of Hepatitis C Virus genotype 4a (HCV4a) as a safe and an efficient vaccine delivery system.</p><p>Methods and results</p><p>Gel.NPs were synthesized and characterized (size: 150±2 nm and zeta potential +17.6 mv). <i>NS2</i> gene was successfully cloned and expressed into E. coli M15 using pQE-30 vector. Antigenicity of the recombinant <i>NS2</i> protein was confirmed by Western blotting to verify the efficiency of <i>NS2</i> as a possible vaccine. Then <i>NS2</i> gene was conjugated to gelatin nanoparticles and a successful conjugation was confirmed by labeling and imaging using Confocal Laser Scanning Microscope (CLSM). Interestingly, the transformation of the conjugated <i>NS2</i>/Gel.NPs complex into E. coli DH5-α was 50% more efficient than transformation with the gene alone. In addition, conjugated <i>NS2</i>/Gel.NPs with ratio 1:100 (w/w) showed higher transformation efficiency into E. coli DH5-α than the other ratios (1:50 and 2:50).</p><p>Conclusion</p><p>Gel.NPs effectively enhanced the gene delivery in bacterial cells without affecting the structure of <i>NS2</i> gene and could be used as a safe, easy, rapid, cost-effective and non-viral vaccine delivery system for HCV.</p></div

Characterization of Gel.NPs.

<p><b>A</b>. HR-TEM image of the prepared Gel.NPs resuspended in water and adsorbed onto solid support and stained with phosphotangestic acid which shows that particles have well dispersed spherical shape with average particles. <b>B</b>. Graph showing particle size distribution by number for the synthesized Gel.NPs that was obtained by particle size analyzer. As shown from the observed peak the size of Gel.NPs is 150±2 nm. C. Graph showing the Zeta potential for synthesized Gel.NPs that was determined by measuring the electrophoretic mobility of the Gel.NPs using a Malvern zeta sizer. As shown the observed Zeta potential is +17.6 mV. D. XRD of prepared GNPs.</p

Cloning and expression of <i>NS2</i> gene in pQE30.

<p><b>A</b>. Agarose gel electrophoresis for single digestion of the miniprep of the recombinant NS2 samples with <i>SphI</i>. Lane M: 1 kb DNA marker, Lane 1–4: DNA resulted from minipreps of four different clones digested with <i>SphI</i> showing a band of ~ 4 kb indicating <i>NS2</i> insert (650 bp) within the pQE-30 vector (3.4 kb). <b>B</b>. Representative DNA sequence analysis of the <i>NS2</i> insert into pQE-30 vector using ABI PRISM model 310 DNA automated sequencer. Bases 1–50 are pQE-30 vector sequence, bases from 50 to the end are partial <i>NS2</i> sequene. Shadowed sequence is the restriction site for <i>SphI</i>. <b>C</b>. <b>a</b>: SDS-PAGE of M15 bacterial lysate resulted from expression of the <i>NS2</i> protein Lane M: prestained wide range MW marker. Lane 1: expressed <i>NS2</i> protein after purification. <b>b</b>: Western blot of the purified <i>NS2</i> protein recognized by sera of Egyptian patients infected with HCV. <b>c</b>: Western blot for healthy donors showed no specific recognition of the <i>NS2</i> protein.</p