A review of RGD-functionalized nonviral gene delivery vectors for cancer therapy.

The development of effective treatments that enable many patients suffering from cancer to be successfully cured is highly demanded. Angiogenesis, which is a process for the formation of new capillary blood vessels, has a crucial role in solid tumor progression and the development of metastasis. Antiangiogenic therapy designed to prevent tumor angiogenesis, thereby arresting the growth or spread of tumors, has emerged as a non-invasive and safe option for cancer treatment. Due to the fact that integrin receptors are overexpressed on the surface of angiogenic endothelial cells, various strategies have been made to develop targeted delivery systems for cancer gene therapy utilizing integrin-targeting peptides with an exposed arginine-glycine-aspartate (RGD) sequence. The aim of this review is to summarize the progress and prospect of RGD-functionalized nonviral vectors toward targeted delivery of genetic materials in order to achieve an efficient therapeutic outcome for cancer gene therapy, including antiangiogenic therapy.

MR traceable delivery of p53 tumor suppressor gene by PEI-functionalized superparamagnetic iron oxide nanoparticles.

Cancer gene therapy involves the replacement of missing or altered genes with healthy ones. In this paper, we have proposed tumor suppressor gene-carrying superparamagnetic iron oxide nanoparticles (SPIONs) for anti-cancer gene therapy. Thermally crosslinked SPIONs (TCL-SPIONs) were conjugated with branched polyethylenimine (PEI 1800 Da) by EDC-NHS chemistry for p53 plasmid DNA delivery. The morphology of the bPEI conjugated TCL-SPIONs (bPEI-TCL-SPION) and pDNA-loaded bPEI-TCL-SPION nanoparticles was measured using transmission electron microscopy (TEM). The particle sizes of the pDNA-loaded bPEI-TCL-SPION nanoparticles were also confirmed by dynamic light scattering, and ranged from 100 to 130 nm, depending on the molar charge ratio. The fluorescently labeled pDNA was complexed with bPEI-TCL-SPION and its intracellular internalization was investigated using confocal microscopy. The p53 plasmid-loaded bPEI-TCL-SPION nanoparticles achieved significantly higher p53 tumor suppressor gene expression and cellular viability compared to positive controls. The expressed wild-type p53 protein suppressed tumor cell proliferation as compared to the mutant control. When transgene expression of the p53 tumor suppressor gene was evaluated at the mRNA level and quantified using real-time PCR, the results were highly dependent on the molar charge ratio (N/P) as well as the cancer cell type. SPIONs internalized within cancer cells were tracked by magnetic resonance (MR) imaging. It was concluded that bPEI-TCL-SPION could be used as efficient gene delivery carriers that can be tracked by MR imaging.

A novel thermosensitive polymer with pH-dependent degradation for drug delivery.

A class of thermosensitive biodegradable multiblock copolymers with acid-labile acetal linkages were synthesized from Pluronic triblock copolymers (Pluronic P85 and P104) and di-(ethylene glycol) divinyl ether. The novel polymers were engineered to form thermogels at body temperature and degrade in an acidic environment. The Pluronic-based acid-labile polymers were characterized using nuclear magnetic resonance, gel permeation chromatography and differential scanning calorimetry. In vitro biocompatibility of the synthesized polymers was evaluated using calorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The polymers showed reverse thermogelling behavior in water around body temperature. The sol-gel transition temperatures of the polymers synthesized from Pluronic P85 and P104 were lowered from 70.3 to 30 degrees C and from 68.5 to 26.9 degrees C, respectively, when the synthesized polymers were compared with corresponding Pluronic block copolymers at a concentration of 25wt.%. The hydrophobic dye solubilization confirmed the formation of polymeric micelles in the aqueous solution. The sizes of the multiblock copolymers increased on a rise in temperature, indicating that thermal gelation was mediated by micellar aggregation. The thermally driven hydrogels showed preferential polymer degradation at acidic pH. At pH 5.0 and 6.5, the release of 40kDa fluorescein isothiocyanate-dextran (FITC-dextran) from the thermally formed hydrogels was completed within 2 and 9 days, respectively. However, FITC-dextran was continuously released up to 30 days at neutral pH. The mechanism of FITC-dextran release at pH 5.0 was mainly an acid-catalyzed degradation, whereas both diffusion and pH-dependent degradation resulted in FITC-dextran release at pH 6.5. The novel polymers hold great potential as a pH-sensitive controlled drug delivery system owing to their interesting phase transition behavior and biocompatibility.

The effects of recombinant human parathyroid hormone, rhPTH(1-84), on bone mass in undernourished rats.

Energy intake restriction reduces bone formation both in protein-energy malnourished children and in undernourished rats, and such conditions might cause partial or irreversible bone loss. Because the use of anti-resorptive agents in this situation is seemingly limited, we examined the effect of the anabolic agent, recombinant human parathyroid hormone (rhPTH(1-84)), on bones in undernourished conditions. First, the osteopenic changes of rat bones with 40% restricted diet for 4 weeks were confirmed. Subsequently, another set of the rats were randomized into four groups and studied for 8 weeks: the freely fed group (control group); the restricted diet, then freely fed group (restriction-ad libitum group); the restricted diet-vehicle-treated group (restriction-vehicle group); and the restricted diet-PTH-treated group (restriction-PTH group). In the restriction-vehicle group, total femoral bone mineral density (BMD) was lower and femoral length was shorter than the control group by 15.4% and 8.1% respectively (P<0.05). In the restriction-ad libitum group, these parameters recovered fully to those of the control group. In the case of intermittent PTH treatment in the persistent undernourished state, the BMD of total femur caught up with those of the control or the restriction-ad libitum group. However, the femoral length remained shorter than those of the other groups. Serum osteocalcin was significantly reduced in continuously undernourished rats, whereas it was elevated in the restriction-PTH group. In conclusion, BMD of total femur was low in undernourished rats. However, it increased after re-feeding ad libitum or intermittent PTH treatment. We suggest that rhPTH(1-84) may be a possible therapeutic agent for ongoing bone loss, especially in patients in a chronically undernourished condition.

Serum leptin, IGF-I and insulin levels in preterm infants receiving parenteral nutrition during the first week of life.

Leptin is involved in the regulation of body weight through a feedback signal between adipose tissue and the satiety center, to decrease food intake and increase energy expenditure. Newborn infants experience physiological weight loss during the first week of life. The leptin level may be decreased to enhance food intake and to decrease energy expenditure for physiological adaptation during early postnatal days. Insulin-like growth factor-I (IGF-I) and insulin are involved in the regulation of perinatal growth. Leptin might be interrelated with IGF-I or insulin, since both of these have adipogenic and somatotropic effects. We therefore hypothesized that leptin, IGF-I and insulin would be decreased during the first week of life, concurrently with physiological weight loss. Thirty preterm AGA infants (birth weight 1.574+/-313 g; GA 31.9+/-2.2 wk) were studied. All infants received parenteral nutrition from the third day after birth. Leptin was significantly decreased during the first week of life, and insulin was significantly increased at day 7 vs. day 1 and day 3. IGF-I did not change during the first week of life. Leptin was positively correlated with body weight (r = 0.368, p<0.01), body mass index (r = 0.267, p<0.05), and serum IGF-I (r = 0.330, p <0.01), but not with serum insulin. The percent of weight reduction during the first week of life was not correlated with the percent of leptin reduction during the first week of life. In conclusion, leptin was significantly decreased and positively correlated with body weight and IGF-I during the first week of life. Changes of leptin and insulin might be related to postnatal adaptation in metabolism, but the exact role of leptin, IGF-I and insulin in postnatal physiological weight loss is not clear.

Factors affecting newborn bone mineral content: in utero effects on newborn bone mineralization.

Several factors have been found recently to have a significant impact on newborn bone mineral content (BMC) and developing fetal bone. Recently we showed that maternal vitamin D deficiency may affect fetal bone mineralization. Korean winter-born newborn infants had extremely low serum 25-hydroxyvitamin D (25-OHD), high serum cross-linked carboxy-terminal telopeptide of type I collagen (ICTP; a bone resorption marker), and markedly lower (8 %) total body BMC than summer-born newborn infants. Infant total body BMC was positively correlated with cord serum 25-OHD and inversely correlated with ICTP, which was also negatively correlated with vitamin D status. In three separate studies on North American neonates we found markedly lower (8-12 %) BMC in summer newborn infants compared with winter newborn infants, the opposite of the findings for Korean neonates. The major reason for the conflicting BMC results might be the markedly different maternal vitamin D status of the North American and Korean subjects. Recently, we found evidence of decreased bone formation rates in infants who were small-for-gestational age (SGA) compared with infants who were appropriate-for-gestational age; we reported reduced BMC, cord serum osteocalcin (a marker of bone formation) and 1,25-dihydroxyvitamin D (the active metabolite of vitamin D), but no alterations in indices of fetal bone collagen metabolism. In theory, reduced utero-placental blood flow in SGA infants may result in reduced transplacental mineral supply and reduced fetal bone formation. Infants of diabetic mothers (IDM) have low BMC at birth, and infant BMC correlated inversely with poor control of diabetes in the mother, specifically first trimester maternal mean capillary blood glucose concentration, implying that factors early in pregnancy might have an effect on fetal BMC. The low BMC in IDM may be related to the decreased transplacental mineral transfer. Cord serum ICTP concentrations were higher in IDM than in control subjects, implying increased intrauterine bone resorption. BMC is consistently increased with increasing body weight and length in infants. Race and gender differences in BMC appear in early life, but not at birth. Ethanol consumption and smoking by the mother during pregnancy affect fetal skeletal development.

Prospective evaluation of perinatal risk factors for cerebral palsy and delayed development in high risk infants.

Prematurity, intrauterine infection and perinatal brain injury have been reported to be significant risk factors of cerebral palsy (CP). We examined the perinatal predictors of cerebral palsy and delayed development (DD) in 184 high risk infants. Thirty-five infants were diagnosed as cerebral palsy and delayed development at 12 months corrected age. Antenatal, intrapartum, and neonatal factors were prospectively evaluated in 2 groups of high risk infants compared with controls; Group A (n = 79), infants weighing less than 2,000 g; Group B (n = 43), infants weighing 2,000 g or more. In univariate analysis, there were no significant antenatal and intrapartum factors associated with cerebral palsy and delayed development in either group. We found that significant postnatal risk factors of CP in group A included sepsis (p = 0.008), BPD (bronchopulmonary dysplasia) (p = 0.028), IVH (intraventricular hemorrhage) (p = 0.042), ventriculomegaly (VM) (p = 0.001) and a longer duration of mechanical ventilation (p = 0.001); while in group B, sepsis (p = 0.047) and neonatal seizure (p = 0.027) were significant risk factors. In multivariate analysis, sepsis in group B was a moderate risk factor of CP (OR (odds ratio) 1.47; 95% CI (confidence interval) 1.02-2.13). In conclusion, neonatal sepsis may contribute to the development of cerebral palsy and delayed development. We suggest that high risk infants who have sepsis should be carefully followed for cerebral palsy and delayed development. The prevention of cerebral palsy may be feasible by decreasing neonatal risk factors such as sepsis during the neonatal period.

Bone mineral content is not reduced despite low vitamin D status in breast milk-fed infants versus cow's milk based formula-fed infants.

The effect of low or borderline vitamin D status on bone mineralization of exclusively breast milk-fed infants has not been studied. The low vitamin D status of Korean breast milk-fed infants may theoretically have adverse effects on bone mineralization. Assuming that bone mineral content (BMC) relates in part to vitamin D status, we hypothesized that serum 25-hydroxyvitamin D (25-OHD) concentration and BMC would be low, and serum osteocalcin concentration high, reflecting active bone turnover, in breast milk- versus formula-fed infants born in the winter. Eighteen breast milk- and 17 formula-fed infants were recruited at ages 2 to 5 months. The BMC of the lumbar1-4 spine region was measured by using dual energy x-ray absorptiometry. The BMC and serum osteocalcin levels were similar for both groups. The serum 25-OHD level was significantly lower in breast milk- than formula-fed infants; 44% of the breast milk group versus 6% of the formula group had serum 25-OHD levels less than 28 nmol/L (11 ng/ml), the lower limit of normal. The BMC did not correlate with the serum 25-OHD level. Thus BMC and serum osteocalcin levels in 2- to 5-month-old infants were not different by type of feeding, despite low vitamin D status in breast milk-fed infants. We speculate that adequate mineral absorption occurs during this period from a predominantly (vitamin D independent) passive transport mechanism.

Low total body bone mineral content and high bone resorption in Korean winter-born versus summer-born newborn infants.

Seasonal differences in newborn total body bone mineral content (TBBMC) have not been studied, particularly in relation to alterations in vitamin D status in winter. In vitamin D deficiency bone resorption may be high and bone mineralization low. Bone resorption may be assessed by serum cross-linked carboxyterminal telopeptide of type I collagen (ICTP) measures. Because vitamin D supplements throughout pregnancy are uncommon in Korea, we hypothesized that in Korean winter newborns, TBBMC is low and serum ICTP high from high bone resorption and low 25-hydroxyvitamin D (25-OHD) compared with those in summer newborns. Seventy-one Korean term infants were studied prospectively in summer (July through September, n = 37) versus winter (January through March, n = 34); TBBMC was measured before 3 days of age by dual-energy x-ray absorptiometry. Significant seasonal differences were found: winter newborns had 6% lower TBBMC (least squares means +/- SD; 86.7 +/- 7.7 gm vs 93.9 +/- 7.8 gm, p = 0.0002), lower cord serum 25-OHD (10.7 +/- 8 nm vs 30 +/- 15 nm, p = 0.0001) and 1,25-dihydroxyvitamin D, and higher ICTP (96.4 +/- 20.3 microg/L vs 74.8 +/- 24 microg/L, p = 0.0002) and calcium than summer newborns. TBBMC correlated with serum 25-OHD (r = 0.243, p = 0.047) and inversely with ICTP (r = -0.333, p = 0.008). We suggest that in Korea low maternal vitamin D status in winter results in marked reduction in newborn TBBMC.

Normal serum indices of bone collagen biosynthesis and degradation in small for gestational age infants.

Serum carboxyterminal propeptide of type I procollagen (PICP) and cross-linked carboxyterminal telopeptide of type I collagen (ICTP), new markers of bone collagen type I biosynthesis and degradation, have not been studied in small for gestational age (SGA) infants. In an earlier study, we found a lower bone mineral content (BMC) and decreased serum osteocalcin in SGA than in appropriate for gestational age (AGA) infants, supporting the thesis that decreased fetal bone formation is a cause of lower BMC in SGA. In view of the role of insulin-like growth factor-I (IGF-I) in the regulation of collagen type I synthesis and degradation, and low serum IGF-I concentrations in SGA infants, we hypothesized that serum PICP would be lower and serum ICTP would be higher in SGA than in AGA infants, reflecting decreased bone collagen type I biosynthesis or enhancement of bone collagen type I degradation in SGA. We studied 19 term SGA and 38 term AGA infants that were matched specifically 1:2 by gestation and birth month. There were no differences between SGA and AGA infants in serum PICP nor ICTP concentrations. Serum ICTP was correlated with osteocalcin and with PICP in SGA infants but not in AGA infants. Thus, serum biochemical indices of bone collagen type I biosynthesis and degradation in term SGA infants are similar to those in term AGA infants. These findings are not consistent with the thesis of altered fetal bone collagen type I biosynthesis or degradation in SGA. We suggest that the reduced bone mineral content in SGA infants is predominantly related to a lower supply of minerals rather than defective regulation of bone collagen type I metabolism.

Similar gastric emptying rates for casein- and whey-predominant formulas in preterm infants.

Casein-predominant infant milk formulas have been speculated to predispose to lactobezoar formation in preterm infants due to delayed gastric emptying. There have been, however, no prospective studies to prove this possibility. In a randomized, double-blinded, prospective study, we tested the hypothesis that preterm infants fed casein-predominant milk formula have slower gastric emptying than infants fed whey-predominant formulas. Twenty preterm infants within the first 4 d of life were randomized to receive either the whey-predominant formula Similac Special Care (whey:casein ratio 60:40) or an experimental casein-predominant formula (whey:casein ratio 18:82). Only the protein composition differed between the two formulas. The infants were fed the assigned study formula until they reached approximately 2200 g body weight when a gastric emptying scan was performed, using the designated study formula mixed with 25 microCi of technetium-99 sulfur colloid. Gastric emptying was followed continuously for 2 h. Gastric emptying at 30, 60, 90, and 120 min was similar between the two study groups. The time for 50% gastric emptying was 64.9 +/- 12.3 min for the infants fed the whey-predominant formula and 56.5 +/- 14.8 min for those fed the casein-predominant formula (p = 0.75). We conclude that the rate of gastric emptying in preterm infants fed casein-predominant formulas is similar to that in those fed whey-predominant formulas.

Low bone mineral content and high serum osteocalcin and 1,25-dihydroxyvitamin D in summer- versus winter-born newborn infants: an early fetal effect?

Seasonal differences in bone mineral indices have not been studied in newborn infants. In adults, indicators of bone metabolism may show seasonal variations. In postneonatal infants and possibly in adults, vitamin D metabolism shows seasonal variations. We hypothesized that in winter-born infants, the bone mineral content is low and serum osteocalcin is high, related to increased bone turnover and high serum 1,25-dihydroxyvitamin D [1,25(OH)2D]. We studied 246 healthy, term appropriate-for-gestation infants in winter (January through March; 140 children) and summer (July through September; 106 children). The bone mineral content (BMC) of the one-third distal radius was measured before 3 days of age by photon absorptiometry. Significant seasonal differences were found: summer-born infants had significantly lower BMC, higher serum osteocalcin and 1,25(OH)2D, and lower serum total calcium than winter-born infants. Seasonal differences in BMC remained significant after adjusting for race and sex. BMC was not correlated with serum biochemical measures. Thus, summer-born newborn infants have low BMC and high serum osteocalcin and 1,25(OH)2D than winter-born infants; these findings are the opposite of adult findings. We suggest that seasonal effects on fetal bone operate especially in early pregnancy (approximately 6 months before birth) resulting in a "phase effect" and opposite findings from later life.

Reduced serum osteocalcin and 1,25-dihydroxyvitamin D concentrations and low bone mineral content in small for gestational age infants: evidence of decreased bone formation rates.

In small for gestational age (SGA) infants, bone mineral content (BMC) is low but the reasons are unclear and the possible relationships between calcium-regulating hormones and BMC have not been studied. We hypothesized that BMC would be lower and concentrations of serum parathyroid hormone and 1,25-dihydroxyvitamin D would be higher at birth in SGA infants than in appropriate for gestational age (AGA) infants. Forty-two term SGA infants and 126 term AGA infants, matched 1:3 specifically by gestation (+/- 1 week) and birth month, were studied prospectively. The BMC of the distal one third of the radius was measured before 3 days of age by photon absorptiometry. The BMC was lower in SGA than in AGA infants. Both SGA and AGA infants had lower BMC in summer or spring than in winter; BMC differences between groups remained significant after adjustment for season (p = 0.0001). Cord serum osteocalcin and 1,25-dihydroxyvitamin D values were lower in SGA than in AGA infants. There were no differences between groups in cord serum levels of intact parathyroid hormone, 25-hydroxyvitamin D, calcium, phosphorus, and magnesium. Relationships were positive between BMC and birth weight and were inverse between BMC and intact parathyroid hormone values. We suggest that reduced uteroplacental blood flow in SGA infants may result in reduced fetal-placental production of 1,25-dihydroxyvitamin D, which results in low BMC and low serum osteocalcin values; fetal serum parathyroid hormone values may be relatively elevated because of reduced placental mineral supply.

Low bone mineral content in summer-born compared with winter-born infants.

Possible seasonal differences in newborn bone mineral content (BMC) have not been studied. Adult studies show seasonal variations with lower BMC in winter versus summer. Assuming that BMC variations may relate in part to vitamin D status, we hypothesized that newborn BMC would be lower in winter than summer. BMC of one third distal radius was measured in 55 healthy term newborns using a single beam photon absorptiometer [coefficient of variation (CV) for phantom standard 2.1%]. Infants were enrolled during summer (July-September, 1988) and winter (January-March, 1989) for a longitudinal nutrition study. Contrary to our hypothesis, there was a 12% lower BMC in summer versus winter (mean +/- SD 75.94 +/- 17.42 vs. 86.55 +/- 17.54 mg/cm, respectively; p = 0.035). The difference remained significant after controlling for possible race and gender effects (p = 0.02). We conclude that BMC is lower in summer- compared with winter-born infants. Since any seasonal effects on fetal bone are presumably related to effects through the mother, we speculate that if maternal vitamin D status influences fetal bone mineralization, the effect (possible sunshine deprivation in winter) may operate especially in early pregnancy, thus resulting in lower BMC, evident at birth in summer.

Effect of tolazoline on persistent hypoxemia in severe hyaline membrane disease.

Ten critically-ill preterm infants with severe hyaline membrane disease received tolazoline because of persistent hypoxemia refractory to the administration of 100% oxygen and mechanical ventilation. Seven infants (70%) responded immediately with an increase in PaO2 greater than or equal to 20 mmHg in the umbilical arterial gas within 60 minutes after bolus infusion (1 to 2 mg/kg) of tolazoline. Twenty-four hours later after the tolazoline infusion, the FiO2 had been decreased from 1.0 to a mean of 0.82 +/- 0.16, and the MAP from 16.5 +/- 1.8 to 15.6 +/- 4.5 cm H2O. Four of 7 infants (57%) who had an immediate response survived, whereas none survived out of 3 infants who failed to respond initially. Three infants experienced relatively severe complications possibly related to tolazoline. There appears to be a place for the use of tolazoline in a severely hypoxemic infant with hyaline membrane disease who is being ventilated, and in whom arterial oxygenation cannot be improved by a further increase in the inspired oxygen concentration or by an alteration of ventilator settings.

Exogenous surfactant replacement therapy of hyaline membrane disease in premature infants.

We conducted a clinical trial to assess whether surfactant-TA given within the first six hours of life could improve oxygenation and reduce the ventilatory support in premature infants with hyaline membrane disease (HMD) during the first 24 hours of life. Eight premature infants with severe HMD requiring ventilation were treated, at a mean age of 2.72 hours, with a single intratracheal instillation of surfactant-TA (120 mg/kg). Arterial oxygenation improved dramatically as reflected by the increase of the a/A PO2 ratio and PaO2 to about 2 times the pretreatment values within 3 hours after surfactant treatment. And thus, oxygen concentrations (FiO2) could be reduced and remained significantly lower than pretreatment values during the first 24 hours after treatment. Infants given surfactant-TA required lower mean airway pressure (MAP) and had a significantly decreased ventilatory index (VI) during the first 24 hours after treatment, which reflect the decreased requirement for ventilatory support. Chest radiograph scores significantly improved within 24 hours after treatment compared with pretreatment scores. In this trial, we found that a single intratracheal dose of surfactant-TA given to infants with HMD resulted in improved respiratory status and radiographic findings during the first 24 hours after treatment.