Investigation of properties affecting controlled release of macromolecules from PLGA microspheres

Poly(lactide-co-glycolide), or PLGA, microspheres offer a widely-studied biodegradable option for controlled release of therapeutics. An array of fabrication methodologies have been developed to produce these microspheres with the capacity to encapsulate therapeutics of various types; and produce microspheres of a wide range of sizes for different methods of delivery. The encapsulation, stability, and release profiles of therapeutic release based on physical and thermodynamic properties has also been studied and modeled to an extent. Much research has been devoted to tailoring formulations for improved therapeutic encapsulation and stability as well as selective release profiles. Despite the breadth of available research on PLGA microspheres, further analysis of fundamental principles regarding the microsphere degradation, formation, and therapeutic encapsulation is necessary. This work aims to examine additional fundamental principles related to PLGA microsphere formation and degradation from solvent-evaporation of preformed polymer. In particular, mapping the development of the acidic microenvironment inside the microsphere during degradation and erosion is discussed. Also, the effect of macromolecule size and conformation is examined with respect to microsphere diameter and PLGA molecular weight. Lastly, the effects of mechanical shearing and protein exposure to aqueous media during microsphere formation are examined. In an effort to better understand the acidic microenvironment development across the microsphere diameter, pH sensitive dye conjugated to protein that undergoes conformational change at different acidic pH values was encapsulated in PLGA microspheres of diameters ranging from 40 µm to 80 µm, and used in conjunction with fluorescence resonance energy transfer to measure the radial pH change in the microspheres. Qualitative analysis of confocal micrographs was used to correlate fluorescence intensity with pH value, and obtain the radial pH across the center of the microsphere. Therapeutic encapsulation and release from polymeric microspheres is governed by an interconnected variety of factors, including the therapeutic itself. The globular protein bovine serum albumin, and the elongated and significantly smaller enzyme, lysozyme, were encapsulated in PLGA microspheres ranging from 40 µm to 80 µm in diameter. The initial surface morphology upon microsphere formation, release profiles, and microsphere erosion characteristics were explored in an effort to better understand the effect of protein size, conformation, and known PLGA interaction on the formation and degradation of PLGA microspheres and macromolecule release, with respect to PLGA molecular weight and microsphere diameter. In addition to PLGA behavior and macromolecule behavior, the effect of mechanical stresses during fabrication was examined. Two similar solvent extraction techniques were compared for the fabrication of albumin loaded microspheres. In particular, the homogeneity of the microspheres as well as capacity to retain encapsulated albumin were compared. This preliminary study paves the way for a more rigorous treatment of the effect of mechanical forces present in popular microsphere fabrication. Several factors affecting protein release from PLGA microspheres are examined herein. The technique explored for spatial resolution of the pH inside the microsphere proved mildly effective in producing a reliable method of mapping microsphere pH changes. However, notable trends with respect to microsphere size, PLGA molecular weight, and microsphere porosity were observed. Proposed methods of improving spatial resolution of the acidic microenvironment are also provided. With respect to microsphere formation, studies showed that albumin and lysozyme had little effect on the internal homogeneity of the microsphere. Rather, ionic interactions with PLGA played a more significant role in the encapsulation and release of each macromolecule. Studies also showed that higher instances of mechanical stress led to less homogeneous microspheres with lower protein encapsulation. This suggests that perhaps instead of or in addition to modifying the microsphere formation formulation, the fabrication technique itself should be more closely considered in achieving homogeneous microspheres with desired loading.

Quichua-Spanish language contact in Salcedo, Ecuador: revisiting media lengua syncretic language practices

The purpose of the current thesis is to develop a better understanding of the interaction between Spanish and Quichua in the Salcedo region and provide more information for the processes that might have given rise to Media Lengua, a ‘mixed’ language comprised of a Quichua grammar and Spanish lexicon. Muysken attributes the formation of Media Lengua to relexification, ruling out any influence from other bilingual phenomena. I argue that the only characteristic that distinguishes Media Lengua from other language contact varieties in central Ecuador is the quantity of the overall Spanish borrowings and not the type of processes that might have been employed by Quichua speakers during the genesis of Media Lengua. The results from the Salcedo data that I have collected show how processes such as adlexification, code-mixing, and structural convergence produce Media Lengua-type sentences, evidence that supports an alternative analysis to Muysken’s relexification hypothesis. Overall, this dissertation is developed around four main objectives: (1) to describe the variation of Spanish loanwords within a bilingual community in Salcedo; (2) to analyze some of the prominent and recent structural changes in Quichua and Spanish; (3) to determine whether Spanish loanword use can be explained by the relationship consultants have with particular social categories; and (4) to analyze the consultants’ language ideologies toward syncretic uses of Spanish and Quichua. Overall, 58% of the content words, 39% of the basic vocabulary, and 50% of the subject pronouns in the Salcedo corpus were derived from Spanish. When compared to Muysken’s description of highlander Quichua in the 1970’s, Spanish loanwords have more than doubled in each category. The overall level of Spanish loanwords in Salcedo Quichua has grown to a level between highlander Quichua in the 1970’s and Media Lengua. Similar to Spanish’s lexical influence in Media Lengua, the increase of Spanish borrowings in today’s rural Quichua can be seen in non-basic and basic vocabularies as well as the subject pronoun system. Significantly, most of the growth has occurred through forms of adlexification i.e., doublets, well-established borrowings, and cultural borrowings, suggesting that ‘ordinary’ lexical borrowing is also capable of producing Media Lengua-type sentences. I approach the second objective by investigating two separate phenomena related to structural convergence. The first examines the complex verbal constructions that have developed in Quichua through Spanish loan translations while the second describes the type of Quichua particles that are attached to Spanish lexemes while speaking Spanish. The calquing of the complex verbal constructions from Spanish were employed when speaking standard Quichua. Since this standard form is typically used by language purists, I argue that their use of calques is a strategy of exploiting the full range of expression from Spanish without incorporating any of the Spanish lexemes which would give the appearance of ‘contamination’. The use of Quichua particles in local varieties of Spanish is a defining characteristic of Quichuacized Spanish, spoken most frequently by women and young children in the community. Although the use of Quichua particles was probably not the main catalyst engendering Media Lengua, I argue that its contribution as a source language to other ‘mixed’ varieties, such as Media Lengua, needs to be accounted for in descriptions of BML genesis. Contrary to Muysken’s representation of relatively ‘unmixed’ Spanish and Quichua as the two source languages of Media Lengua, I propose that local varieties of Spanish might have already been ‘mixed’ to a large degree before Media Lengua was created. The third objective attempts to draw a relationship between particular social variables and the use of Spanish loanwords. Whisker Boxplots and ANOVAs were used to determine which social group, if any, have been introducing new Spanish borrowings into the bilingual communities in Salcedo. Specifically, I controlled for age, education, native language, urban migration, and gender. The results indicate that none of the groups in each of the five social variables indicate higher or lower loanword use. The implication of these results are twofold: (a) when lexical borrowing occurs, it is immediately adopted as the community-wide norm and spoken by members from different backgrounds and generations, or (b) this level of Spanish borrowing (58%) is not a recent phenomenon. The fourth and final objective draws on my ethnographic research that addresses the attitudes of syncretic language use. I observed that Quichuacized Spanish and Hispanicized Quichua are highly stigmatized varieties spoken by the country’s most marginalized populations and families, yet within the community, syncretic ways of speaking are in fact the norm. It was shown that there exists a range of different linguistic definitions for ‘Chaupi Lengua’ and other syncretic language practices as well as many contrasting connotations, most of which were negative. One theme that emerged from the interviews was that speaking syncretic varieties of Quichua weakened the consultant’s claim to an indigenous identity. The linguistic and social data presented in this dissertation supports an alternative view to Muysken’s relexification hypothesis, one that has the advantage of operating with well-precedented linguistic processes and which is actually observable in the present-day Salcedo area. The results from the study on lexical borrowing are significant because they demonstrate how a dynamic bilingual speech community has gradually diversified their Quichua lexicon under intense pressure to shift toward Spanish. They also show that Hispanicized Quichua (Quichua with heavy lexical borrowing) clearly arose from adlexification and prolonged lexical borrowing, and is one of at least six identifiable speech styles found in Salcedo. These results challenge particular interpretations of language contact outcomes, such as, ones that depict sources languages as discrete and ‘unmixed.’ The bilingual continuum presented in this thesis shows on the one hand, the range of speech styles that are accessible to different speakers, and on the other hand, the overlapping, syncretic features that are shared among the different registers and language varieties. It was observed that syncretic speech styles in Salcedo are employed by different consultants in varied interactional contexts, and in turn, produce different evaluations by other fellow community members. In the current dissertation, I challenge the claim that relexification and Media Lengua-type sentences develop in isolation and without the influence of other bilingual phenomena. Based on Muysken's Media Lengua example sentences and the speech styles from the Salcedo corpus, I argue that Media Lengua may have arisen as an institutionalized variant of the highly mixed "middle ground" within the range of the Salcedo bilingual continuum discussed above. Such syncretic forms of Spanish and Quichua strongly resemble Media Lengua sentences in Muysken’s research, and therefore demonstrate how its development could have occurred through several different language contact processes and not only through relexification.