PLGA microsphere-mediated growth hormone release hormone expression induces intergenerational growth.

To improve animal growth, growth hormone-releasing hormone (GHRH) expression vectors that maintain constant GHRH expression can be directly injected into muscles. To deliver the GHRH expression vectors, biodegradable microspheres have been used as a sustained release system. Although administering GHRH through microspheres is a common practice, the intergenerational effects of this delivery system are unknown. To investigate the intergenerational effects of polylactic-co-glycolic acid (PLGA) encapsulated plasmid-mediated GHRH supplements, pCMV-Rep-GHRH microspheres were injected into pregnant mice. Growth and expression of GHRH were measured in the offspring. RT-PCR and immunohistochemistry reveal GHRH expression 3-21 days post-injection. The proportion of GH-positive cells in the GHRH treated offspring was 48.2% higher than in the control group (P < 0.01). The GHRH treated offspring were 6.15% (P < 0.05) larger than the control offspring. At day 49 post-injection, IGF-I serum levels were significantly higher in the treatment group than in the control group. This study confirms that intramuscular expression of GHRH mediated by PLGA microspheres significantly enhances intergenerational growth.

Overexpression of GRF encapsulated in PLGA microspheres in animal skeletal muscle induces body weight gain.

Biodegradable nanospheres or microspheres have been widely used as a sustained release system for the delivery of bioagents. In the present study, injectable sustained-release growth hormone-releasing factor (GRF) (1-32) microspheres were prepared by a double emulsion-in liquid evaporation process using biodegradable polylactic-co-glycolic acid (PLGA) as the carrier. The entrapment efficiency was 89.79% and the mean particle size was 4.41 mum. The microspheres were injected into mouse tibialis muscle. After 30 days, mice injected with GRF (1-32) microspheres (group I) gained significantly more weight than any other treatment group, including mice injected with the naked plasmid (group II) (10.26 +/- 0.13 vs. 9.09 +/- 0.56; P < 0.05), a mixture of microspheres and plasmid (group III) (10.26 +/- 0.13 vs. 8.57 +/- 0.02; P < 0.05), or saline (IV) (10.26 +/- 0.13 vs. 6.47 +/- 0.26; P < 0.05). In addition, mice treated with the GRF (1-32) microspheres exhibited the highest expression levels of GRF as detected by PCR, RT-PCR, and ELISA (mean 2.56 +/- 0.40, P < 0.05, overall comparison of treatment with groups II, III, and IV). Additionally, rabbits were injected in the tibialis muscle with the same treatments described above. After 30 days, the group treated with GRF (1-32) microspheres gained the most weight. At day 30 postinjection, weight gain in group I was 63.93% higher than group II (plasmid) (877.10 +/- 24.42 vs. 535.05 +/- 26.38; P < 0.05), 108.59% higher than group III (blank MS) (877.10 +/- 24.42 vs. 420.50 +/- 19.39; P < 0.05), and 93.94% higher than group IV (saline) (877.10 +/- 24.42 vs. 452.25 +/- 27.38; P < 0.05). Furthermore, IGF-1 levels in the serum from GRF microsphere-treated group were elevated relative to all other groups. The present results suggest that encapsulation of GRF with PLGA increases GRF gene expression in muscle after local plasmid delivery, and stimulates significantly more weight gain than delivery of the naked plasmid alone.

[The expression of growth hormone release factor gene carried with PLGA microspheres in muscle tissue and its effect on mouse growth].

Injectable sustained-release pcDNA3-GRF (1-32) microspheres were prepared by double emulsion-in liquid evaporation process,using biodegrable poly lactic-co-glycolic acid as carrier. The enrapment efficiency, mean particle size, drug content thus prepared were 69%, 2.20 microm, 8% and 70% respectively. The result of transfection in vivo showed that after 30 days, accumulative increased body weights on the group injected with pcDNA3-GRF (1-32) microspheres was significantly higher than those group injected with naked plasmid (12.87%), plasmid-empty microspheres (19.72%) and saline (58.58%) respectively. PCR and RT-PCR showed that the expression level of GRF gene on the group injected with pcDNA3-GRF (1-32) microspheres was the highest. GRF gene released by microspheres was still detected after 30 days. In conclusion, pcDNA3-GRF (1-32) microspheres have a controlled release effect and GRF gene could be successfully transfected into muscle cells of mouse by microspheres with higher efficacy and stronger biological function.

Alpha-helical assembly of biologically active peptides and designed helix bundle protein.

The formation of alpha-helical assembly by complexing biologically active peptides with de novo designed protein is described. The de novo designed protein described here is a cystine-linked 4-helix bundle protein constructed with 80 amino acid residues and forms a hydrophobic core region surrounded by 4 helices in an aqueous solution. The biologically active peptides, such as melittin and human growth hormone releasing factor, contain the sequences that are able to form amphiphilic helices. These peptides alone do not form the alpha-helix structure in a diluted solution with low ion strength. But on mixing with the designed helix bundle protein, the peptides are strongly bound to the protein with the induction of alpha-helical structure in the biologically active peptides. The content of induced alpha-helix is in accord with that estimated from the amphiphilic sequence. The results mean that a novel architecture composed of alpha-helices is formed. Fluorescent and temperature-scanning measurement revealed that the alpha-helical assembly is constructed with hydrophobic interaction. Also, it is shown by means of fluorescence depolarization that the assembly has a compact globular form corresponding to 1:1 complex.