Nanoparticle mediated increased insulin-like growth factor 1 expression enhances human placenta syncytium function

Abstract

Introduction: Placental dysfunction is an underlying cause of many major obstetric diseases and treatment options for complications like fetal growth restriction (FGR) are limited .We previously demonstrated nanoparticle delivery of the human insulin-like growth factor 1 (hIGF1) transgene under control of the trophoblast-specific PLAC1 promoter maintains normal fetal growth in a surgically-induced FGR mouse model. However, uptake by human placental syncytiotrophoblast has yet to be determined.

Methods: An ex vivo human placenta perfusion model, term placenta villous fragments, and other in vitro syncytiotrophoblast models were used to determine nanoparticle uptake, transgene expression, and functional responses under oxidative stress conditions.

Results: In the ex vivo perfusion, fluorescence from a Texas-Red conjugated nanoparticle increased in maternal perfusate upon nanoparticle addition and declined by the conclusion of the experiment (P < 0.001. Fluorescent histology confirmed localization in the syncytiotrophoblasts. No Texas-Red fluorescence was detected in the fetal perfusate. Transgene expression of hIGF1 in differentiated BeWo cells, isolated primary trophoblasts and fragments was increased compared to untreated (55,000-fold, P = 0.0003; 95-fold, P = 0.003; 400-fold, P < 0.001, respectively). Functionally, increased hIGF1 expression in villous fragments resulted in translocation of glucose transporter 1 to the syncytiotrophoblast cell membrane and under conditions of oxidative stress in BeWo cells, protected against increased cell death (P < 0.01) and decreased mitochondrial activity (P < 0.01).

Conclusion: The current study confirms that our nanoparticle is capable of uptake in human placental syncytium which results in enhanced transgene expression, functional changes to cellular activity and protection against increased oxidative stress.

Keywords: Fetal growth restriction; Insulin-like growth factor; Nanoparticle; Placenta; Placental dysfunction; Pregnancy; Therapy.

Figure 1. Analysis of Texas-red fluorescence in maternal and fetal perfusate samples, and placental tissue from the dual placental perfusion experiments.

Prior to addition of Texas-red conjugated nanoparticle (NP) to maternal perfusate, no Texas-red fluorescence was recorded in maternal perfusate (A). Upon addition of nanoparticle, Texas-red fluorescence increased in maternal perfusate and declined after approximately 1 hour of perfusion. Texas-red fluorescence was not determined in fetal perfusate at any of the time points (A). Histological analysis of placental tissue collected at the conclusion of the experiment showed nanoparticle localization in the syncytiotrophoblast layer of the fetal villi (B negative control tissue: perfused tissue with no Texas-red conjugated nanoparticle added, C positive tissue: tissue perfused with Texas-red conjugated nanoparticle for approximately 1 hour). Fluorescence analysis of the tissue sections confirmed a significant increase in Texas-red in the tissue sections exposed to nanoparticle (D). Data are mean ± SEM, n=7 term placentas perfused; n=6 term placentas perfused with NP. Statistical significance determined with an ANOVA and Tukey’s post hoc analysis (A) and Mann-Whitney test (D). Scale bar = 50 μm.

Figure 2. qPCR analysis of human insulin-like growth factors 1 (hIGF1) mRNA expression in differentiated BeWo and isolated human placental syncytiotrophoblasts after nanoparticle treatment.

Forskolin treatment of BeWo cells for 24 hours induced differentiation and formation of syncytial-like cells (A and B). qPCR analysis of hIGF1 expression in differentiated BeWo cells showed no difference in mRNA expression in untreated (control), forskolin treated (FK), nor forskolin + plasmid alone treated (FK+Plac1-hIGF1) cells, however, a significant increase in hIGF1 mRNA expression was found in the forskolin + hIGF1 nanoparticle (FK+NP-Plac1-hIGF1) treated cells (C). Similarly in isolated syncytiotrophoblast cells, treatment with plasmid alone (Plac1-hIGF1) did not increase hIGF1 mRNA expression compared to untreated (control) but nanoparticle (NP-Plac1-hIGF1) treatment resulted in a significant increase (D). Data are median ± interquartile range, n=6 passages (C) and n=4 isolated placentas (D). Statistical significance was determined with a Kruskal-Wallace test and Dunn’s post hoc analysis.

Figure 3. qPCR analysis of human insulin-like 1 (hIGF1) mRNA expression and immunohistochemical (IHC) localization of glucose transporter 1 (SLC2A1) in placenta fragments treated with nanoparticle.

qPCR analysis of hIGF1 mRNA showed a significant increase in placenta fragments treated with nanoparticle (NP-Plac1-hIGF1) at 24 hours which was sustained at 72 hours (A). There was no increase in hIGF1 mRNA expression in fragments treated with nanoparticle containing a plasmid encoding the green fluorescent protein gene (NP-Plac1-GFP)(A). In situ hybridization confirmed plasmid specific mRNA expression of IGF1 in syncytiotrophoblast cells of fragments 72 h after treatment with NP-Plac1-hIGF1 and not in untreated fragments (B). Representative images of IHC staining of SLC2A1 in fragments at 48 hours showed trans-localization of the transporter to the apical (closed triangle) and basal (open triangle) membranes of the syncytiotrophoblast and cytotrophoblasts cells in the fragments treated with nanoparticle (NP-Plac1-hIGF1) compared to untreated (control) (C). Data are median ± interquartile range, n=6 term placentas. Scale Bar: 200 μm (C top row), 50 μm (B and C middle row), 10 μm (C bottom row). Statistical significance was determined with a 2-way ANOVA.

Figure 4. Functional effect of nanoparticle treatment in protecting against oxidative stress in BeWo cells.

qPCR analysis of human insulin-like growth factor 1 (hIGF1) was increased in cells treated with nanoparticle (NP-Plac1-hIGF1 and H₂O₂+NP-Plac1-hIGF1) compared to untreated (control) and H₂O₂ alone (A). Compared to untreated, treatment with H₂O₂ significantly increased the percentage of dead cells but not when cells were pretreated with nanoparticle (B). Cell number was lower in cells treated with H₂O₂ alone and nanoparticle with H₂O₂ compared to untreated and nanoparticle alone treated (C). The BAX:BCL2 ratio was higher in cells treated with H₂O₂ as well as in those treated with nanoparticle and H₂O₂ when compared to untreated and nanoparticle alone treated (D). Mitochondrial activity, as measured by the percentage of rezaurin reduction, was lower in cells treated with H₂O₂ alone compared to untreated but not in cells treated with nanoparticle prior to H₂O₂ treatment (E). Data are median ± interquartile range (A) or mean ± SEM (B-E), n=6 passages. Statistical significance was determined with a repeated measures ANOVA and Sidak’s multiple comparison test.