影响PLGA微球包封率和粒径相关因素研究进展

聚乳酸乙醇酸共聚物(PLGA)是一种可生物降解的高分子聚合物,具有良好的生物相容性,其降解产物为乳酸和乙醇酸,是机体正常代谢的中间产物,最终可分解为二氧化碳和水,并分别经肺和肾脏排出体外,对人体不产生危害,所以PLGA在微球制剂的制备中常作为首选载体。近年来PLGA微球制剂在医药领域有着飞跃发展,尤其是在抗肿瘤、免疫疫苗、蛋白给药、基因治疗、诊断试剂和细胞支架等方面显现出很大优势。而且已有许多PLGA微球获得美国FDA批准上市,临床应用也有令人满意的效果,未见报道有严重的不良反应。但现阶段国内生产的PLGA缓释微球的质量还有很多不足之处如微球粒径偏大、包封率和载药量偏低、突释过大等,有待进一步提高和改进。本文将综述在制备包裹水溶性药物的PLGA微球过程中相关因素如药物本身理化性质、制备方法、PLGA结构特点、有机溶剂等对微球粒径、包封率的影响,以期为提高以PLGA为药物载体的药效奠定良好的理论基础。     

Efficiency Considerations for Group Sequential Designs with Adaptive Unblinded Sample Size Re-assessment

Clinical trials with adaptive sample size re-assessment, based on an analysis of the unblinded interim results (ubSSR), have gained in popularity due to uncertainty regarding the value of \(\delta \) at which to power the trial at the start of the study. While the statistical methodology for controlling the type-1 error of such designs is well established, there remain concerns that conventional group sequential designs with no ubSSR can accomplish the same goals with greater efficiency. The precise manner in which this efficiency comparison can be objectified has been difficult to quantify, however. In this paper, we present a methodology for making this comparison in a standard, well-accepted manner by plotting the unconditional power curves of the two approaches while holding constant their expected sample size, at each value of \(\delta \) in the range of interest. It is seen that under reasonable decision rules for increasing sample size (conservative promising zones, and no more than a 50% increase in sample size) there is little or no loss of efficiency for the adaptive designs in terms of unconditional power. The two approaches, however, have very different conditional power profiles. More generally, a methodology has been provided for comparing any design with ubSSR relative to a comparable group sequential design with no ubSSR, so one can determine whether the efficiency loss, if any, of the ubSSR design is offset by the advantages it confers for re-powering the study at the time of the interim analysis.     

Relationship Between Cell Size and Efficiency of Synchronization During Nitrogen-Limited Phased Cultivation of Candida utilis

Under the phased method of cultivation the yeast Candida utilis grew and divided synchronously. The newly formed cells were relatively small, and a new cell cycle was not initiated until the cells could attain a certain minimum size (critical size). Although the cells expanded to some extent after division, the critical size was not reached until a fresh supply of medium was provided. With the arrival of the fresh supply of growth medium at the beginning of the phasing period, the cells expanded rapidly, and new cell cycles were initiated. The cells continued to expand until the growth-limiting nutrient (nitrogen source) was exhausted or until 90 min, which ever occurred first. Usually, buds emerged at a constant time after the start of the phasing period. The time of bud emergence was independent of the size attained by the cells during the expansion phase of growth. The results indicated that it was initiation of the cell cycle that was under size control, and not bud emergence. Bud emergence seemed to be under the control of a timer. The start of this timer seemed to be at or immediately after the beginning of the phasing period. Protein synthesis was essential for the initiation and expansion of buds. However, inhibition of protein synthesis by cycloheximide did not prevent unbudded cells or the parent portion of budded cells from expanding. Cycloheximide seemed to abolish the control mechanism(s) which prevented the cells from expanding after they had reached the maximum size.     

Electrospun Chitosan Microspheres for Complete Encapsulation of Anionic Proteins: Controlling Particle Size and Encapsulation Efficiency

Electrospinning was employed to fabricate chitosan microspheres by a single-step encapsulation of proteins without organic solvents. Chitosan in acetic acid was electrospun toward a grounded sodium carbonate solution at various electric potential and feeding rates. Electrospun microspheres became insoluble and solidified in the sodium carbonate solution by neutralization of chitosan acetate. When the freeze-dried microspheres were examined by scanning electron microscopy, the small particle size was obtained at higher voltages. This is explained by the chitosan droplet size at the electrospinning needle was clearly controllable by the electric potential. The recovery yield of chitosan microspheres was dependent on the concentration of chitosan solution due to the viscosity is the major factor affecting formation of chitosan droplet during curling of the electrospinning jets. For protein encapsulation, fluorescently labeled bovine serum albumin (BSA) was codissolved with chitosan in the solution and electrospun. At higher concentration of sodium carbonate solution and longer solidification time in the solution, the encapsulation efficiency of the protein was confirmed to be significantly high. The high encapsulation efficiency was achievable by instant solidification of microspheres and electrostatic interactions between chitosan and BSA. Release profiles of BSA from the microspheres showed that the protein release was faster in acidic solution due to dissolution of chitosan. Reversed-phase chromatography of the released fractions confirmed that exposure of BSA to acidic solution during the electrospinning did not result in structural changes of the encapsulated protein.     

Focus on Water: Stomatal Size,Speed, and Responsiveness Impact on Photosynthesis and Water Use Efficiency

The control of gaseous exchange between the leaf and bulk atmosphere by stomata governs CO₂ uptake for photosynthesis and transpiration, determining plant productivity and water use efficiency. The balance between these two processes depends on stomatal responses to environmental and internal cues and the synchrony of stomatal behavior relative to mesophyll demands for CO₂. Here we examine the rapidity of stomatal responses with attention to their relationship to photosynthetic CO₂ uptake and the consequences for water use. We discuss the influence of anatomical characteristics on the velocity of changes in stomatal conductance and explore the potential for manipulating the physical as well as physiological characteristics of stomatal guard cells in order to accelerate stomatal movements in synchrony with mesophyll CO₂ demand and to improve water use efficiency without substantial cost to photosynthetic carbon fixation. We conclude that manipulating guard cell transport and metabolism is just as, if not more likely to yield useful benefits as manipulations of their physical and anatomical characteristics. Achieving these benefits should be greatly facilitated by quantitative systems analysis that connects directly the molecular properties of the guard cells to their function in the field.In order for plants to function efficiently, they must balance gaseous exchange between inside and outside the leaf to maximize CO₂ uptake for photosynthetic carbon assimilation (A) and to minimize water loss through transpiration. Stomata are the “gatekeepers” responsible for all gaseous diffusion, and they adjust to both internal and external environmental stimuli governing CO₂ uptake and water loss. The pathway for CO₂ uptake from the bulk atmosphere to the site of fixation is determined by a series of diffusional resistances, which start with the layer of air immediately surrounding the leaf (the boundary layer). Stomatal pores provide a major resistance to flux from the atmosphere to the substomatal cavity within the leaf. Further resistance is encountered by CO₂ across the aqueous and lipid boundaries into the mesophyll cell and chloroplasts (mesophyll resistance). Water leaving the leaf largely follows the same pathway in reverse, but without the mesophyll resistance component. Guard cells surround the stomatal pore. They increase or decrease in volume in response to external and internal stimuli, and the resulting changes in guard cell shape adjust stomatal aperture and thereby affect the flux of gases between the leaf internal environment and the bulk atmosphere. Stomatal behavior, therefore, controls the volume of CO₂ entering the intercellular air spaces of the leaf for photosynthesis. It also plays a key role in minimizing the amount of water lost. Transpiration, by virtue of the concentration differences, is an order of magnitude greater than CO₂ uptake, which is an inevitable consequence of free diffusion across this pathway. Although the cumulative area of stomatal pores only represents a small fraction of the leaf surface, typically less than 3%, some 98% of all CO₂ taken up and water lost passes through these pores. When fully open, they can mediate a rate of evaporation equivalent to one-half that of a wet surface of the same area (Willmer and Fricker, 1996).Early experiments illustrated that photosynthetic rates were correlated with stomatal conductance (g_s) when other factors were not limiting (Wong et al., 1979). Low g_s limits assimilation rate by restricting CO₂ diffusion into the leaf, which, when integrated over the growing season, will influence the carbohydrate status of the leaf with consequences for crop yield. Stomata of well-watered plants are thought to reduce photosynthetic rates by about 20% in most C3 species and by less in C4 plants in the field (Farquhar and Sharkey, 1982; Jones, 1987). However, even this restriction has been shown to impact substantially on yield. For example, Fischer et al. (1998) demonstrated a close correlation between g_s and yield in eight different wheat (Triticum aestivum) cultivars. Those studies highlighted the effects g_s can have on crop yield, not only through reduced CO₂ diffusion but also through the impact on water loss and evaporative cooling of the leaf. Indeed, enhancing photosynthesis yields by only 2% to 3% is sufficient to substantially increase plant growth and biomass over the course of a growing season (Lefebvre et al., 2005; Zhu et al., 2007).Stomata and their behavior profoundly affect the global fluxes of CO₂ and water, with an estimated 300 × 10¹⁵ g of CO₂ and 35 × 10¹⁸ g of water vapor passing through stomata of leaves every year (Hetherington and Woodward, 2003). Changes in stomatal behavior in response to changing climatic conditions are thought to impact on water levels and fluxes. For example, it is estimated that partial stomatal closure driven by increasing CO₂ concentration over the past two decades has led to increased CO₂ uptake and reduced evapotranspiration in temperate and boreal northern hemisphere forests (Keenan et al., 2013), with implications for continental runoff and freshwater availability associated with the global rise in CO₂ (Gedney et al., 2006). Concurrently, the increase in global water usage over the past 100 years and the expectation that this is set to double before 2030 (UNESCO, 2009) has put pressure on breeders and scientists to find new crop varieties, breeding traits, or potential targets for manipulation that would result in crop plants that are able to sustain yield with less water input. The fact that stomata are major players in plant water use and the entire global water cycle makes the functional and physical attributes of stomata potential targets for manipulation to improve carbon gain and plant productivity as well as global water fluxes.There are several approaches for improving carbon gain and plant water use efficiency (WUE) that focus on stomata. It is possible to increase or decrease the gaseous conductance of the ensemble of stomata per unit of leaf area (g_s) through the manipulation of stomatal densities (Büssis et al., 2006). In addition, there is potential to alter the stomatal response or sensitivity to environmental signals through the manipulation of guard cell characteristics that affect stomatal mechanics (e.g. OPEN STOMATA [ost] mutants; Merlot et al., 2002). Such approaches have produced an array of mutant plants with altered characteristics and varying impacts on CO₂ uptake and transpiration, several of which we discuss in greater detail below. An intuitive measure of the efficacy of such manipulations is the WUE, commonly defined as the amount of carbon fixed in photosynthesis per unit of water transpired. In general, higher WUE values have been observed in plants with lower g_s, but these gains are usually achieved together with a reduction in A and slower plant growth. Plants with higher g_s have greater assimilation rates and grow faster under optimal conditions, but they generally exhibit lower WUE. An approach that has not been fully explored or considered in any depth is to select plants for differences in the kinetics of stomatal response or to manipulate stomatal kinetics in ways that improve the synchrony with mesophyll CO₂ demand (Lawson et al., 2010, 2012). To date, the majority of studies assessing the impact of stomatal behavior on photosynthetic carbon gain have focused on steady-state measurements of g_s in relation to photosynthesis. These studies do not take account of the dynamic situation in the field. As we discuss below, a cursory analysis of stomatal synchrony with mesophyll CO₂ demand suggests that gains of 20% to 30% are theoretically possible.Here, we address the question of the kinetics of the stomatal response to the naturally fluctuating environment, notably to fluctuations in light that are typical of the conditions experienced in the field. We focus on vascular seed plants, to which crop plants belong, and do not address seedless vascular plants, such as ferns. The characteristics of the latter, and hence the issues and challenges they present, are very different. In our minds, of paramount importance is whether there is potential for engineering guard cells of crop plants to manipulate the dynamic behavior of stomata so as to improve WUE without substantial cost in assimilation. Of course, in many circumstances, stomata are not the only factor to limit water flux through the plant (see other articles in this issue), but they are one of the most important “gatekeepers” and therefore, serve as a good starting point for such considerations. Thus, we explore the physical and functional attributes of stomata, their signaling, and the solute transport mechanisms that determine pore aperture as targets for potential manipulation of stomatal responses to changing environmental cues.     

Effects of Insert Size on Transposition Efficiency of the Sleeping Beauty Transposon in Mouse Cells

Transposon vectors are widely used in prokaryotic and lower eukaryotic systems. However, they were not available for use in vertebrate animals until the recent reconstitution of a synthetic fish transposon, Sleeping Beauty (SB). The reacquisition of transposability of the SB transposase fostered great enthusiasm for using transposon vectors as tools in vertebrate animals, particularly for gene transfer to facilitate accelerated integration of transgenes into chromosomes. Here, we report the effects of insert sizes on transposition efficiency of SB. A significant effect of insert size on efficiency of transposition by SB was found. The SB transposase enhanced the integration efficiency effectively for SB transposon up to approximately 5.6 kb, but lost its ability to enhance the integration efficiency when the transposon size was increased to 9.1 kb. This result indicates that the SB transposon system is highly applicable for transferring small genes, but may not be applicable for transferring very large genes. Received October 20, 2000; accepted December 15, 2000.     

Task Partitioning in Insect Societies. I. Effect of Colony Size on Queueing Delay and Colony Ergonomic Efficiency

The collection and handling of colony resources such as food, water, and nest construction material is often divided into subtasks in which the material is passed from one worker to another. This is known as task partitioning. When material is transferred directly from one individual to another, queueing delays frequently occur because individuals must sometimes wait for a transfer partner. A stochastic simulation model was written to study the effect of colony size on these delays. Queueing delay decreases roughly exponentially with colony size because stochastic fluctuations in the arrival of individuals are lower in larger colonies. These results support empirical studies of Polybia occidentalis and other theoretical studies of honeybees. The effect of the relative number of individuals in the two subtask groups was also studied. There is a unique optimal ratio of the number of workers associated with each of the subtasks that simultaneously minimizes mean queueing delay and maximizes colony nectar-processing rate. Deviations from this optimal ratio, for example, as a result of forager mortality or changes in nectar productivity that affect foraging trip duration, increase mean queueing delays greatly, especially in smaller colonies.     

Neocortex Size Predicts Group Size in Carnivores and Some Insectivores

Neocortex size has been shown to correlate with group size in primates. Data for carnivores and insectivores are used to test the generality of this relationship. The data suggest that carnivores lie on the same grade as the primates, but that insectivores lie on a separate grade to the left of these two orders. Among the insectivores, there appears to be a distinction between the ‘advanced’ genera (which show a relationship between group size and neocortex size) and the ‘basal’ genera (which do not).     

Size Matters: Individual Variation in Ectotherm Growth and Asymptotic Size

Body size, and, by extension, growth has impacts on physiology, survival, attainment of sexual maturity, fecundity, generation time, and population dynamics, especially in ectotherm animals that often exhibit extensive growth following attainment of sexual maturity. Frequently, growth is analyzed at the population level, providing useful population mean growth parameters but ignoring individual variation that is also of ecological and evolutionary significance. Our long-term study of Lake Erie Watersnakes, Nerodia sipedon insularum, provides data sufficient for a detailed analysis of population and individual growth. We describe population mean growth separately for males and females based on size of known age individuals (847 captures of 769 males, 748 captures of 684 females) and annual growth increments of individuals of unknown age (1,152 males, 730 females). We characterize individual variation in asymptotic size based on repeated measurements of 69 males and 71 females that were each captured in five to nine different years. The most striking result of our analyses is that asymptotic size varies dramatically among individuals, ranging from 631–820 mm snout-vent length in males and from 835–1125 mm in females. Because female fecundity increases with increasing body size, we explore the impact of individual variation in asymptotic size on lifetime reproductive success using a range of realistic estimates of annual survival. When all females commence reproduction at the same age, lifetime reproductive success is greatest for females with greater asymptotic size regardless of annual survival. But when reproduction is delayed in females with greater asymptotic size, lifetime reproductive success is greatest for females with lower asymptotic size when annual survival is low. Possible causes of individual variation in asymptotic size, including individual- and cohort-specific variation in size at birth and early growth, warrant further investigation.     

The Effect of Nonideal Cascade Impactor Stage Collection Efficiency Curves on the Interpretation of the Size of Inhaler-Generated Aerosols

Cascade impactors, operating on the principle of inertial size separation in (ideally) laminar flow, are used to determine aerodynamic particle size distributions (APSDs) of orally inhaled product (OIP) aerosols because aerodynamic diameter can be related to respiratory tract deposition. Each stage is assumed typically to be an ideal size fractionator. Thus, all particles larger than a certain size are considered collected and all finer particles are treated as penetrating to the next stage (a step function stage efficiency curve). In reality, the collection efficiency of a stage smoothly increases with particle size as an “S-shaped” curve, from approximately 0% to 100%. Consequently, in some cases substantial overlap occurs between neighboring stages. The potential for bias associated with the step-function assumption has been explored, taking full resolution and two-stage abbreviated forms of the Andersen eight-stage nonviable impactor (ACI) and the next-generation pharmaceutical impactor (NGI) as example apparatuses. The behavior of unimodal, log-normal APSDs typical of OIP-generated aerosols has been investigated, comparing known input values to calculated values of central tendency (mass median aerodynamic diameter) and spread (geometric standard deviation, GSD). These calculations show that the error introduced by the step change assumption is larger for the ACI than for the NGI. However, the error is sufficiently small to be inconsequential unless the APSD in nearly monodisperse (GSD ≤1.2), a condition that is unlikely to occur with realistic OIPs. Account may need to be taken of this source of bias only for the most accurate work with abbreviated ACI systems.     

Statistical and Economical Efficiency in Assessment of Liver Regeneration Using Defined Sample Size and Selection in Combination With a Fully Automated Image Analysis System

Quantification of liver regeneration is frequently based on determining the 5-bromo-2-deoxyuridine labeling index (BrdU-LI). The quantitative result is influenced by preanalytical, analytical, and postanalytical variables such as the region of interest (ROI). We aimed to present our newly developed and validated automatic computer-based image analysis system (AnalySIS-Macro), and to standardize the selection and sample size of ROIs. Images from BrdU-labeled and immunohistochemically stained liver sections were analyzed conventionally and with the newly developed AnalySIS-Macro and used for validation of the system. Automatic quantification correlated well with the manual counting result (r=0.9976). Validation of our AnalySIS-Macro revealed its high sensitivity (>90%) and specificity. The BrdU-LI ranged from 11% to 57% within the same liver (32.96 ± 11.94%), reflecting the highly variable spatial distribution of hepatocyte proliferation. At least 2000 hepatocytes (10 images at 200× magnification) per lobe were required as sample size for achieving a representative BrdU-LI. Furthermore, the number of pericentral areas should be equal to that of periportal areas. The combination of our AnalySIS-Macro with rules for the selection and size of ROIs represents an accurate, sensitive, specific, and efficient diagnostic tool for the determination of the BrdU-LI and the spatial distribution of proliferating hepatocytes. (J Histochem Cytochem 57:1075–1085, 2009)     

Egg-Mass Size and Cell Size: Effects of Temperature on Oxygen Distribution

Two processes strongly influence the distribution of oxygenwithin egg masses and cells: the supply of oxygen by diffusionand the consumption of oxygen by embryos and mitochondria. Theseprocesses are differentially sensitive to temperature. The diffusioncoefficient of oxygen depends only weakly on temperature, havinga Q₁₀ of approximately 1.4. In contrast, the consumption ofoxygen depends strongly on temperature, having a Q₁₀ between1.5 and 4.0. Thus, at higher temperatures, the ratio of oxygensupply to demand decreases. I show, by extending a model ofoxygen distribution within metabolizing spheres, that maximalegg-mass sizes and cell sizes are predicted to be smaller athigher temperatures. For egg masses, definitive data are notyet available. For ectothermic cells, this prediction appearsto be supported; cells from a variety of ectothermic organisms,unicellular and multicellular, are smaller when the cells areproduced at warmer temperatures. Establishing a specific connectionbetween this pattern and oxygen distributions requires demonstrationof (1) oxygen concentration gradients within metabolizing spheresand (2) central oxygen concentrations low enough to affect function.Egg masses from a variety of taxa show steep oxygen concentrationgradients and often are severely hypoxic or anoxic in centrallocations. Severe hypoxia appears capable of retarding developmentor killing embryos. Similar kinds of data for ectothermic cellshave not yet been collected, but the literature on oxygen gradientswithin mammalian cells suggests that intracellular gradientsmay be important.     

Subcellular Size

All of the same conceptual questions about size in organisms apply equally at the level of single cells. What determines the size, not only of the whole cell, but of all of its parts? What ensures that subcellular components are properly proportioned relative to the whole cell? How does alteration in organelle size affect biochemical function? Answering such fundamental questions requires us to understand how the size of individual organelles and other cellular structures is determined. Knowledge of organelle biogenesis and dynamics has advanced rapidly in recent years. Does this knowledge give us enough information to formulate reasonable models for organelle size control, or are we still missing something?