Low Transfer of Tacrolimus and Its Metabolites into Colostrum of Graft Recipient Mothers

Currently, the majority of neonates born to organ recipient mothers on chronic immunosuppressive therapy are formula fed. However, over the past few years, evidence has grown, suggesting that breastfeeding might be possible and beneficial. We designed a study assessing the transfer of tacrolimus into the colostrum of posttransplant mothers. We assessed the amount of tacrolimus and its metabolites, M-1 and M-3, that would be ingested by the breastfed neonates. Concentrations of tacrolimus and its metabolites were measured in colostrum from 14 posttransplant mothers as well as in venous cord blood and venous blood of the neonates. Test material analysis was performed by liquid chromatography coupled with mass spectrometry (LC/MS). The amount of ingested formula was registered, which allowed for estimation of the amount of tacrolimus and its metabolites that would be ingested by breastfed infants. The mean amount of tacrolimus that would be ingested by the neonates in maternal milk was 151.4 ng/kg/24 h (standard deviation SD � 74.39); metabolite M-1: 23.80 ng/kg/24 h (SD � 14.53); and metabolite M-3: 13.25 ng/kg/24 h (SD � 9.05). The peak level of tacrolimus and metabolite M-1 in colostrum was noted 8 h after an oral dose (3.219 ng/mL SD � 2.22 and 0.56 ng/mL SD � 0.60, respectively) and metabolite M-3 after 6 h (0.29 ng/mL SD � 0.22). Low concentrations of tacrolimus and its metabolites, M-1 and M-3, in colostrum show that neonates will ingest trace amounts of the drug. Further studies are required to fully assess the safety of breastfeeding by posttransplant mothers

Purification of recombinant adenovirus type 3 dodecahedric virus-like particles for biomedical applications using short monolithic columns.

Adenovirus type 3 dodecahedric virus-like particles (Ad3 VLP) are an interesting delivery vector. They penetrate animal cells in culture very efficiently and up to 300,000 Ad3 VLP can be observed in one cell. The purification of such particles usually consists of several steps. In these work we describe the method development and optimization for the purification of Ad3 VLP using the Convective Interaction Media analytical columns (CIMac). Results obtained with the CIMac were compared to the already established two-step purification protocol for Ad3 VLP based on sucrose density gradient ultracentifugation and the Q-Sepharose ion-exchange column. Pure, concentrated and bioactive VLP were obtained and characterized by several analytical methods. The recovery of the Ad3 VLP was more than 50% and the purified fraction was almost completely depleted of DNA; less than 1% of DNA was present. The purification protocol was shortened from five days to one day and remarkably high penetration efficacy of the CIMac-purified vector was retained. Additionally, CIMac QA analytical column has proven to be applicable for the final and in-process control of various Ad3 VLP sample

Virus-like particle-mediated intracellular delivery of mRNA cap analog with in vivo activity against hepatocellular carcinoma.

Adenovirus dodecahedron (Dd), a nanoparticulate proteinaceous biodegradable virus-like particle (VLP), was used as a vector for delivery of an oncogene inhibitor to hepatocellular carcinoma (HCC) rat orthotopic model. Initiation factor eIF4E is an oncogene with elevated expression in human cancers. Cell-impermeant eIF4E inhibitor, cap structure analog (cap) and anti-cancer antibiotic doxorubicin (Dox) were delivered as Dd conjugates. Dd-cap and Dd-dox inhibited cancer cell culture proliferation up to 50 and 84%, respectively, while with free Dox similar results could be obtained only at a 5 times higher concentration. In animal HCC model the combination treatment of Dd-cap/Dd-dox caused 40% inhibition of tumor growth. Importantly, the level of two pro-oncogenes, eIF4E and c-myc, was significantly diminished in tumor sections of treated rats. Attachment to Dd, a virus-like particle, permitted the first demonstration of cap analog intracellular delivery and resulted in improved doxorubicin delivery leading to statistically significant inhibition of HCC tumor growth

Adenovirus dodecahedron, as a drug delivery vector.

Stability studies show that Dds can be conveniently stored and transported, and can potentially be used for therapeutic purposes under various climates. Successful BLM delivery by Ad Dds demonstrates that the use of virus like particle (VLP) results in significantly improved drug bioavailability. These experiments open new vistas for delivery of non-permeant labile drugs

Kinetics of ds DNA breaks as jugded by induction of γ-H2AX foci.

<p>HeLa cells were treated either with Dd, with free BLM or Dd-BLM for indicated periods and analyzed with anti-γ-H2AX Ab (in red) and with anti-tubulin Ab (in green) by confocal microscopy, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>. Scale bar equals 10 µm.</p

Purification of recombinant Ad3 DB expressed in the baculovirus system.

<p>(A) Dodecahedra initially purified on sucrose density gradient were fractionated on a Q-Sepharose column in 20 mM Tris buffer, pH 7.5, using NaCl gradient. (B) Analysis of purified Dds. Left panel - negative stain electron microscopy (EM) of Dds purified on sucrose density gradient. Middle and right panels - non-denaturing agarose gel electrophoresis of fractions recovered from the Q-Sepharose column with detection with ethidium bromide (EtBr, middle panel) followed by Commassie Brilliant Blue (CBB) staining (right panel). (C) Negative stain EM showing free pentameric bases recovered in peak 1 (left panel) and complete dodecahedra in peak 2 (right panel) of the Q-Sepharose column (P1 and P2 in A). Scale bar equals 100 nm. (D) Flow cytometry analysis of HeLa cells transduced with Dd (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>). Sucrose density gradient-purified Dds – purple curve, Q-Sepharose-purified Dds - green curve. Blue curve shows the antibody background in the absence of Dd.</p

Dd stability upon lyophilization, inside HeLa cells and in human serum.

<p>(A) Purified Dds were dialyzed overnight at 4°C against water or 150 mM (NH₄)₂SO₄ in water. Mannitol (0.4%) and sucrose (0.4%) were added to samples marked „Cryoprotect. +”. Dd samples were frozen at −80°C, dried in speed-vac or lyophilized (marked Lyoph. +). Dry samples were reconstituted in the starting volume of water. All preparations were centrifuged for 30 min at 13000 rpm and the supernatants were applied onto native agarose gel. (B) Stability of Dd after application to HeLa cells. Purified Dds (2 µg in 100 µl) were applied to 2×10⁴ portions of HeLa cells. After indicated periods of penetration cell lysates were analyzed on SDS-PAGE (left panel) or on native agarose gel (two right panels). Control Dd samples contained 30 ng protein, while control Pb sample contained 10 ng protein. (C) Stability of Dd upon incubation in human serum. Samples of Dd concentrated by ultrafiltration in Microcon unit (Millipore) (5 µg each) were incubated in human serum (HS) at 4°C for 2 h (lane 4) and at 37°C for 15 min or for 2 h (lanes 5 and 6, respectively). Samples were resolved by native agarose gel electrophoresis and analyzed by Western blot performed with anti-Dd serum. The upper part shows CBB stained gel with proteins remaining after transfer, and the lower part the developed Western blot. Lanes 1 and 7 show Dd non treated or incubated for 2 h at 37°C, respectively, in the absence of serum. Lanes 2 and 3 show human serum after 2 h incubation at 4 and 37°C, respectively. Dd samples incubated with the serum are denoted in bold.</p

Effect of Dd and Dd-BLM conjugate on HeLa cells.

<p>(A) Flow cytometry analysis of Dd (green curve) and Dd-BLM (pink curve) cell entry. Cells were treated with appropriate vector for 1 h at 37°C as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>. The blue curve shows the antibody background in the absence of Dd. (B) Cells were treated with 1 µg Dd or Dd-BLM for indicated times and analyzed with anti-Dd serum (in red) by confocal microscopy, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>. Nuclei were stained blue with DAPI. Last row shows the 50 h-treatment without nuclear staining. Scale bar equals 20 µm.</p

Cell toxicity of bleomycin (BLM) delivered with the aid of Dd.

<p>BLM was chemically attached to Dds as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>. (A) Characterization of Dd-BLM conjugate by mass spectrometry analysis (Maldi). (B) DLS analysis of the BLM conjugate. (C) MTT assay of cell toxicity. HeLa cells were treated with free BLM (0.13 µM), Dd (1 µg) and Dd-BLM (1 µg delivering 0.08 µM BLM), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>.</p

Thermal Dd stability as a function of pH and ionic strength.

<p>(A) Dd was analyzed by dynamic light scattering (DLS) at different pH in the presence of 150 mM NaCl as a function of temperature, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005569#s4" target="_blank">Material and Methods</a>. (B) Native gel analysis of Dds and Pbs, in CAPS pH 9, and carbonate buffer, pH 10. Some samples were subjected to temperature treatment imitating DLS temperature gradient (marked DLS). (C) DLS analysis carried out on Dds in PBS under different ionic strength conditions. Mean values of three apparatus readings are shown.</p