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Bud Spencer. Jesse Eisenberg. Da Younow Deutsch auch Marit. Dominic Rtl Ninja Warrior. Terrence Howard. Naomie Harris. Als dann der Musiker Christian in die Familie kommt, staunt Vor Einbruch Der Nacht Jonas nicht schlecht Schocktherapie Im Zentrum der ungewöhnlichen Familiengeschichte stehen Benno Fürmann und Maja Schöne als glückliches Paar mit zwei Kindern, das auch ohne Trauschein fest an sein Zusammensein glaubt und sich daher auf eine unkonventionelle Abmachung Die Rückkehr Des Königs Extended Stream kann. Johannes Herrschmann. Richard Griffiths. However, it is cautioned that formulation of FA as one of theses forms may lead to interconversion to Form III in the presence of an aqueous milieu potentially resulting in a reduction in the release rate of the drug. This website uses cookies to improve your experience while you navigate through the website. Degree of crystallinity determinations were not carried out in this thesis, and will not be described further. Using a modified approach, Gupta et al. Said Senhaji — Moulay Ahmed. Refworks Account Login. The nature of the polymer-drug dispersion Freddy’S New Nightmare been shown to play a significant role in the performance Naima Kelm the drug delivery system Leuner and Dressman, Thus, our group has proposed that a biodegradable polymer-based microsphere system could potentially be co-located at the implant site to release Crazy Rich Asians Stream Deutsch loaded antibiotics, would not require surgical removal, and may be an appropriate delivery system to combat prosthetic joint infections. Of particular importance in pharmaceutical systems are the thermodynamic differences, Podemus generally exist between any set of polymorphic pairs. Reda Rais — Khsara Fik.

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Examples of homopolymers are poly lactic acid and poly glycolic acid. However, if the polymer is made of two or more different monomers A and B , the polymer is referred to as a copolymer.

Copolymers may be subdivided into four main classifications Figure 1. The properties of a polymer differ greatly depending on its classification.

For example, a theoretical block copolymer composed of monomer A and monomer B, will have properties of both homopolymer A and homopolymer B.

However, if A and B are randomly distributed, or alternating along the polymer backbone, the polymer will have properties intermediate between homopolymer A and homopolymer B, and dependent on the relative contributions of monomer A and monomer B in the final polymer.

In this thesis, we utilize poly D,L-lactic acid-co-glycolic acid PLGA , with a LA:GA ratio of , which is a random copolymer composed of equal amounts of lactic acid and glycolic acid.

The physical properties of PLGA are discussed in section 1. Since polymers do not achieve equilibrium in the solid state, the partial ordered or disordered arrangement between chains gives rise to semi- crystalline and amorphous material, respectively Rosen, Semi-crystalline polymers contain regions of crystallinity, or crystallites, distributed throughout amorphous polymer chains Seymour and Carraher, In this model, polymer crystallites, or fringed micelles, are composed of regularly ordered areas of aligned segments of polymer chains interspersed in an amorphous matrix.

Since the polymer backbones are many times longer than the crystallites, the polymer chains may pass from one crystallite to another through amorphous regions of randomly arranged polymer chains Seymour and Carraher, Due to the random nature of the polymerization process, no two polymer chains grow equally fast or for the same duration of time, and therefore, there exists a distribution of molecular weights in any polymer material Cowie, The molecular weight of a polymer is expressed as the average molecular weight of the entire molecular weight distribution.

The molecular weight distribution of polymers is characterized by different average molecular weights, including the number averaged molecular weight!

The number averaged molecular weight! This average is sensitive to the presence of very small chains since all molecules contribute equally to the average value Rosen, A small fraction of the polymer sample, by weight, may exist as small polymer chains or low molecular weight impurities, yet this fraction may represent a large proportion of the total number of molecules present in the polymer, skewing!

The weight averaged molecular weight! This average is less sensitive to the presence of short chains in a 39 polymer sample. For instance, for!

The lower sensitivity of! Molecular Weight W eig ht F ra cti on 40 1. Therefore, molecular weight is an important determinant of a number of physical properties including glass transition temperature and melt temperature.

The glass transition temperature Tg of a polymer is defined as the onset of large-scale cooperative motion, due to rotational and translational movement, of polymer chain segments in the order of consecutive carbon atoms upon heating Ebewele, The Tg is affected by the polymer molecular weight according to the following equation, derived by Fox and Flory Fox and Flory, : Equation 1.

Therefore, an increase in polymer molecular weight results in a proportional increase in polymer Tg. Semi-crystalline polymers contain small crystallites dispersed throughout an amorphous matrix.

The melting of these crystallites is considerably different from the melting behavior of small molecules, as they usually melt over a broad range, and are subject to melting point depression due to the presence of small molecule diluents termed plasticizers , such as solvent molecules, which may originate due to the method of crystallization Mandelkern, As previously mentioned, polymers melt over a broad range of temperatures, reflecting both a range in the nature of crystallites within the matrix and in the molecular weights of polymer chains in polydisperse materials Mandelkern, A number of controlled release systems based on polymers have been developed to treat a wide range of disease states such as drug-eluting stents for the treatment of restenosis Regar et al.

In addition, a myriad of drugs have been incorporated into polymeric microspheres see section 1. In the reservoir system, a drug phase with or without polymer is surrounded by a rate-limiting polymer phase, and in a matrix system, the drug is uniformly dispersed within a polymer Figure 1.

In this thesis we focus our efforts in the development of matrix-type drug delivery systems for the treatment of implant-associated infections.

Natural polymers, such as polysaccharides and proteins, belong to the latter class of enzymatically-sensitive polymers, whereas synthetic polymers, such as the aliphatic polyesters, polyanhydrides, poly ortho esters , belong to the former.

In general, the most widely used synthetic class of polymers is the aliphatic polyesters, which includes poly glycolic acid , poly lactic acid , poly D,L-lactic acid-co-glycolic acid and poly -caprolactone.

These polyesters offer the attractiveness of low cost, their reproducibility in manufacturing, their biocompatibility and lack of toxicity with prolonged use Vert, Although the degree of crystallinity is limited, it can be controlled to some extent through the inclusion of stereoregular monomers, and annealing.

Upon exposure to aqueous milieu, the ester carbon in PLGA is subject to nucleophilic attack by water molecules according to Scheme 1. In addition, the carboxylic acid end groups produced from the hydrolytic degradation of PLGA ester bonds are able to catalyze the hydrolysis of other ester groups, a process called autocatalysis Gopferich, Therefore, the degradation of PLGA occurs heterogeneously throughout the matrix, with rates increasing towards the center of the device von Burkersroda et al.

R groups represent CH3 for lactide, and H for glycolide. Zhou et al. Increasing the GA ratio of the copolymer has been shown to influence water uptake, resulting in faster degradation rates due to preferential degradation of GA units Li, ; Wu and Wang, In addition, increasing the molar content of stereoregular LA can increase the overall matrix crystallinity Chye Joachim Loo et al.

However, since PLGA is primarily amorphous, matrix crystallinity is not expected to contribute to overall degradation rate.

The molecular weight of PLGA has also been shown to be a significant factor in the rate of degradation and mass loss. Wu and Wang, have shown that decreasing the!

The longer polymer chains in polymers with higher! However, higher molecular weight polymers do not exhibit any obvious morphology change until their molecular weight is lowered enough to lose mechanical integrity because of their high molecular weight Wu and Wang, Although the degradation rates are faster, the erosion of matrices composed of higher molecular weight polymers is slower.

In this thesis, we use PLGA with a molecular weight of 49, Da, which shows significant mass loss i. The breakdown of PLGA to oligomers or constitutive monomers allows it to be cleared by endogenous metabolic pathways: LA is oxidized to pyruvate or converted to glucose via gluconeogenesis where it is subsequently cleared from the body through the Krebs or Cori cycles, respectively, and GA is excreted unchanged in the urine Vert et al.

D dCdx Equation 1. In drug delivery systems where a drug is dissolved and molecularly dispersed within the matrix, it is called a monolithic solution.

In monolithic 47 solutions there initially exists no concentration gradient to drive drug transport. However, as surface drug is depleted a concentration gradient is produced allowing drug to diffuse down the concentration gradient, through the polymer to the surface and is gradually released at the surface Baker, Where drug is not molecularly dispersed but present as solid drug particles throughout the polymer matrix due to drug insolubility in the polymer matrix or a drug loading that exceeds the drugs solubility in the polymer, it is defined as a monolithic dispersion Baker, The dispersion can be classified as simple, complex, or as a monolithic matrix system and is based on the amount of drug loaded into the polymer matrix.

In these systems, solid drug particles are well dispersed and separated and release involves dissolution of drug particles in the polymer free volume followed by diffusion through polymer chains to the surface of the matrix.

Release from simple monolithic devices can be described by the simple Higuchi model as follows Higuchi, : Equation 1. Release from a simple monolithic device will be linear when plotted as a function of the square root of time.

Release of drugs from complex monolithic dispersions is still proportional to the square root of time, however have a higher rate than predicted from simple Higuchi kinetics.

Release from monolithic matrices can be described by the percolation theory, where drug release occurs by dissolution of the active ingredient and diffusion through water-filled capillaries created from the dissolution of interconnecting drug particle clusters, creating a porous network Holman and Leuenberger, In these systems drug must first partition into the water-filled pores, and thus the diffusion coefficient of the drug through the polymer no longer describes the release profile.

The presence of pores, and porosity of the matrix ' significantly increases the release of drug from monolithic matrices, and is defined by the second form of the Higuchi equation Higuchi, , which incorporates both and : Equation 1.

In many polymeric, controlled-release formulations, an initial large bolus of drug is released when introduced to the release medium, prior to the onset of diffusion controlled and polymer degradation and erosion mediated drug release.

The burst phase is diffusion rate limited, and is the release of drug that is closely associated with the surface of the device. There are many factors that contribute to the burst phase of drug release, including drug-polymer compatibility see section 1.

However, the primary factors influencing the burst phase are drug loading, surface area, and polymer molecular weight.

Curve A represents drug release from a device with a significant burst phase of drug release Allison, ; curve B represents drug release from monolithic matrices where no burst phase is observed Baker, ; curve C represents drug release from granular matrices Higuchi, ; and curve D represents drug release from matrices undergoing bulk erosion over the course of drug release, where the onset of polymer erosion causes significant drug release from the polymeric Baker, In addition to diffusional transport, polymer degradation rate also influences drug release, as shown in Figure 1.

In bulk degradation, as seen in PLGA matrices, water influx into the polymer matrix is faster than the breakdown of the matrix into smaller, water soluble components Sinko and Kohn, Therefore, hydrolysis of susceptible polymer bonds i.

The pores and channels formed from the hydrolysis of polymer chains into soluble oligomers and monomers leads to erosion and subsequent mass loss of the polymeric device Figure 1.

These factors can be categorized into polymer properties, including molecular weight, monomer ratios, end-group capping, and crystallinity; drug properties, including physicochemical characteristics, extent of loading, and distribution throughout the polymer DrugPolymer Drug Remaining Polymer Voids Due To Polymer Erosion 52 matrix; and device characteristics, including size, porosity, density, and shape Fredenberg et al.

This section will focus on the major factors influencing drug release from PLGA matrices. Since these matrices are primarily amorphous, factors such as crystallinity will not be discussed further.

The influences of monomer ratio and molecular weight have been shown to be strong regulators of matrix erosion, and drug release from biodegradable matrices Witt et al.

For PLGA, increasing the lactide content has been associated with a decrease in drug release rate for a number of drugs, including nalbuphine Sung et al.

The increased lactide content in PLGA reduces matrix hydrophilicity, slows matrix hydration and delays erosion Zhou et al.

The physicochemical properties of the drug can also play a role in regulating drug release. Miyajima et al.

In addition to solubility, the compatibility between drug and polymer see section 1. The geometry of the drug delivery system is another predictor of drug release.

Crank Crank, , and Cobby Cobby et al. This phenomenon has been supported through experimental observations of drug release from microspheres see section 1.

In fact, an alteration in processing to produce a porous device has been shown to markedly improve drug release. Klose et al.

The influence of a number of these factors on the drug release from PLGA microspheres and nanofibers will be reviewed in sections 1. The nature of the polymer-drug dispersion has been shown to play a significant role in the performance of the drug delivery system Leuner and Dressman, When drug is present as a molecular level dispersion within a carrier, it is also termed a solid solution.

Solid solutions are comparable to liquid solutions, consisting of a single phase irrespective of the number of molecules or components Leuner and Dressman, Solid solutions can be classified according to miscibility, and may be described as a continuous or discontinuous solid solution Leuner and Dressman, This suggests that the bonding strength between the individual components is stronger than the bonding strength 54 between molecules of their own kind.

No continuous solid solutions have been reported in the peer-reviewed drug delivery literature Leuner and Dressman, A typical phase diagram of a discontinuous solid solution is shown in Figure 1.

Increasing the amount of one of these components above its solubility limit produces a system characterized by 2 phases. Solid solutions of sulfathiazole and urea are classic examples of a discontinuous solid solution Chiou and Niazi, In amorphous solid solutions, the drug molecules are dispersed on a molecular level throughout the amorphous polymer matrix.

Amorphous solid solutions are a single phase, characterized by a reduction in polymer Tg due to the plasticizing effects of the drug molecules.

In semi-crystalline polymers, where some degree of crystallinity exists, drug may also partition into spaces between polymer chains in crystallites, and be characterized by a lower Tg and a melting point depression.

Amorphous solid solutions were first described by Chiou, who used citric acid to increase the solubility of griseofulvin Chiou and Riegelman, Other examples have been illustrated by van Drooge who used thermal analysis to illustrate the interactions of diazepam with PVP.

Amorphous suspensions contain two distinct phases: one characteristic of the polymer, and one characteristic of the drug. Solid suspensions occur when there is a reduced solubility of the drug in the polymer Chiou and Riegelman, For example, Yang et al.

When formulated as a PLGA microsphere using solvent evaporation techniques see section 1. In chapter 3 of this thesis, we expand these previous observations made by our group, and characterize a PLGA microsphere formulation containing FA and RIF as an amorphous solid suspension of drug rich domains dispersed throughout a polymer matrix.

However, XRD is unable to discriminate between amorphous solid solutions and amorphous solid suspensions. Additional techniques, such as Raman spectroscopy, and thermal analysis allow for the collection of more useful information, allowing for proper categorization of the dispersed system Chiou and Riegelman, ; van Drooge et al.

The dispersion of a drug in a polymeric carrier has been shown to markedly improve the dissolution and release profile of many hydrophobic drugs including, griseofulvin Chiou and Riegelman, , itraconazole Jung et al.

Therefore, the nature of drug-polymer dispersions is important when designing and evaluating drug delivery devices.

Whether a polymeric drug delivery device will form a solution or suspension i. In order for a solute to be solubilized by a solvent in this case a drug solute molecule solubilized in the polymer solvent matrix , the intermolecular forces between the solute and solvent must be greater than solvent-solvent and solute-solute interactions.

The amount of energy necessary to overcome the solute-solute, and solvent-solvent interactions must be greater than the cohesive energy between molecules in each of the two phases.

In other words, the cohesive energy Ecoh is the amount of energy required to remove a unit volume per mole of molecules from their neighboring molecules into infinite separation Bicerano, The amount of energy required to do so in a unit volume of material V is defined as the cohesive energy density ecoh , such that: Equation 1.

Therefore, indirect methods, such as comparative swelling, or dissolution in liquids of known Ecoh, may yield values of Ecoh for non-evaporating material Bicerano, However, in , Dunkel showed that the Ecoh for low molecular weight organic compounds is additive, and can be predicted by summing the contributions of each molecular group constituting the overall compound Dunkel, The cohesive forces between materials are a result of three intermolecular forces: dispersion forces atomic , polar forces molecular , and hydrogen bonding electron exchange Van Krevelen, This induced polarization dipole , attracts temporary dipoles in neighboring molecules, resulting in cohesion.

In contrast to dipole moments, polar cohesive forces are due to permanent dipoles in neighboring molecules, which arise as a result of electrostatic interactions between electronegative and electropositive atoms between molecules.

Hydrogen bonding is similar to polar cohesive forces, and is the interaction between an electronegative atom of one molecule and an electropositive hydrogen bonded to a nitrogen, oxygen, or fluorine atom on a neighboring molecule.

It is described as a strong electrostatic dipole-dipole interaction, but has features of covalent bonding. Therefore the Ecoh of any molecule must be the sum of the individual energies that make it up, and is defined by van Krevelen as: Equation 1.

The calculation of Ecoh, thus far, has been used in the context of vaporization. That is the energy required to overcome cohesive forces between like molecules.

The free energy change for the solution process is given by: Equation 1. In the case of polymer solutions, Flory Flory, and Huggins Huggins, expanded on observations from Fried, who experimentally determined that predictions for ideal solutions were not always appropriate when dealing with molecules with larger molecular sizes such as polymers Fried, The Flory-Huggins interaction parameter sp was developed!

The Flory-Huggins solution theory states that the sp is always positive, and is proportional to the enthalpy of mixing. The molecular-level drug effectively plasticizes the polymer by increasing the free volume and subsequently lowers the polymer Tg.

Therefore, a miscible blend of two amorphous components where one component is solubilized by the other, in this case a compatible drug-polymer pair, will exhibit a single Tg that will vary with the composition.

However, the most commonly employed equation is the Fox equation Fox, , which is defined as: Equation 1.

The critical tenet of systemic antibiotics is that there exists a relationship between plasma drug concentrations and drug concentrations at the surgical site.

In fact, for infections of extracellular compartments, this has been evaluated for a number of antibiotic classes.

In these studies, tissue penetration has been related with plasma protein binding Bergan et al. However, these predictions only represent the diffusional transport of drug into extracellular tissue fluid.

Antibiotic penetration into abscesses and sub-cutaneous fibrin clots has been shown to be considerably slower than into extracellular fluid compartments Barza and Weinstein, Therefore, the limitation to successful antimicrobial therapy is an inability to reach adequate tissue levels due to the threshold of systemic toxicity.

The delivery of the drug locally, to the infection site, may circumvent this limitation. There are a number of proposed techniques for the local administration of antibiotics to prevent and treat infections associated with surgical implants.

These include, low pressure irrigation with an antimicrobial solution Bhandari et al. PMMA is a nondegradable polymer Figure 1. The major liquid components are methylmethacrylate monomer, benzoyl peroxide initiator, hydroquinone as a stabilizer to prevent premature polymerization, and a green chlorophyll dye, and the principle powder components are high molecular weight PMMA microbeads, barium sulphate or zirconium dioxide as radio-opacifiers, and a green chlorophyll dye, which helps to distinguish between cement and bone in the final mixture Webb and Spencer, ; Jaeblon, The liquid and powder components are combined and mixed to form a malleable grout, which is then used to fix implantation devices.

In a two-stage revision surgery, where the primary implant fails, PMMA has been manually formed into spacers of similar shape to the prosthetic, which are used to occupy boney voids to promote tissue healing prior to the implantation of a new sterile prosthetic.

It is during the mixing stage that antibiotics are introduced into the bone cement for use as an antibiotic eluting device. There has been widespread adoption of antibiotic-loaded bone cements ALBCs among surgeons in the United States for a number of years Heck et al.

Until recently, the widespread adoption of ALBC in North America has been done off-label, through the mixing of sterile antibiotic in polymerizing PMMA bone cements in the operating theatre, as no current guidelines exist for the use of ALBCs as a prophylactic.

These ALBC products are only approved for use in the second stage, of a two-stage revision surgery where initial infection was cleared see section 1.

Although there are a number of studies suggesting that there is sufficient evidence for the use of ALBCs as a method for prophylaxis or treatment of infection, there exists much debate over its widespread use Bourne, ; Bourne, ; Hanssen, The major concerns regarding the use of ALBCs are due to the possible promotion of antibiotic resistance through drug release profiles that lead to sub-MIC at the surgical site.

Due to the impermeability of PMMA, antibiotic release from acrylic bone cements is a surface phenomenon, and is almost entirely dependent on the exposed surface area van de Belt et al.

The sub-therapeutic elution has been a concern for the development of antibiotic resistance. In fact, in , Hope et al.

Furthermore, Dunne et al. These data suggest that the resistance of clinical isolates to gentamicin should preclude the use of ALBCs containing gentamicin.

In fact, due to the emerging resistance of bacteria to aminoglycoside antibiotics, a number of groups have suggested the incorporation of a secondary antibiotic, including fusidic acid Neut et al.

However, Anagnostakos et al. Investigations of the tensile strength, hardness and flexion of ALBCs has been thoroughly investigated. Failure of the bone cement matrix can lead to aseptic loosening and implant failure via macrophage-mediated inflammatory response, which recruits osteoclasts to 68 the cement-bone interface, resulting in bone resorption Horowitz and Purdon, In almost all cases, ALBCs had reductions in mechanical strength, which was dependent on the type and amount of antibiotic used De Palma et al.

In addition to bacterial growth, the thermal curing of PMMA in situ may also lead to the degradation of antibiotics and local tissue damage.

The collagen sponge is the most widely used product of the protein-based materials, and has been evaluated for the localized delivery of gentamicin, cefotaxime, fusidic acid, clindamycin and vancomycin Wachol-Drewek et al.

In all cases, the drugs eluted from collagen sponge over 4d, with the majority of drug released within the first 24 h.

Gentamicin- 69 collagen sheets have been shown to be a clinically effective treatment in intramedullary nailing Hettfleisch and Schottle, , periprosthetic breast implant infections Lapid, , and in the treatment of osteomyelitis Ipsen et al.

However, some physicians suggest that further refinement is needed before it should be used clinically, which is likely one of the reasons why it is not approved for use in the US McLaren, Polymeric implants have been used in many forms, including thin films for periodontal applications Agarwal et al.

Microspheres loaded with cefazolin have been particularly successful in treating infections associated with tibial fractures in animal models when placed in the surgical pocket immediately following surgery, prior to wound closure Jacob et al.

Thus, our group has proposed that a biodegradable polymer-based microsphere system could potentially be co-located at the implant site to release the loaded antibiotics, would not require surgical removal, and may be an appropriate delivery system to combat prosthetic joint infections.

This method consists of four major steps: 1 drug solubilization in an organic solvent containing polymer, creating the dispersed phase; 2 dispersion of the organic phase as droplets in the continuous phase an aqueous phase containing a small amount of surfactant using high sheer stresses imparted by an overhead propeller stirring at high rpm; 3 the diffusion of solvent from dispersed phase into continuous phase, and subsequent evaporation; 4 washing, recovery, and drying of microspheres.

The major determinants of drug release from biodegradable microspheres include, polymer molecular weight and crystallinity, microsphere diameter, and drug loading.

The release of drug from polymeric microspheres can be controlled by the molecular weight of the polymer. Makino et al. Furthermore, the release from high molecular weight polymers was incomplete until the onset of erosion and mass loss, and therefore it was suggested that that the higher molecular weight may have caused a decrease in drug diffusion through the matrix due to decreased matrix hydration.

Microsphere diameter has a pronounced influence on drug release rates Siepmann et al. The smaller microspheres will have a larger proportion of drug on, or near the microsphere surface, and thus release is usually characterized by a larger burst phase of drug release.

Drug loading has also been shown to significantly increase the rate of drug release from PLGA microspheres for a number of drugs including pactlitaxel Mu and Feng, , fentanyl Choi et al.

As shown in section 1. Increasing the drug loading increases the initial concentration in the device leading to a more rapid rate of release down the concentration gradient.

Increasing drug loading can also influence the release of drug due to an alteration of the nature of the drug dispersion e. As discussed in section 1.

Thus, the increased drug release observed with an increase in drug loading may also be due to the increased porosity of the polymer matrix through the formation of water-filled pores and channels created from the dissolution and loss of drug from the polymer matrix.

A report from Faisant et al. These properties have been shown to be favorable in orthopaedic applications, as nanofibers have been used in the coating of medical implants to create a nanoporous matrix in which cells would preferentially adhere and enhance biointegration Badami et al.

They have been used as a hemostatic due to high surface area and absorptive capacities Zhang et al. Furthermore, nanofiber membranes are flexible and can adhere to complex contours, which are often present in operative surfaces.

These electrospun matrices can also serve as drug delivery vehicles through direct incorporation of active therapeutics in the polymer fiber structure, either within a matrix fiber, or within a fiber core see section 1.

Accordingly, nanofibers have been used as drug delivery vehicles to deliver a number of low molecular weight antibiotics including cephazolin Katti et al.

Therefore, our group has suggested that highly flexible nanofiber membranes loaded with antimicrobials may possess favorable characteristics appropriate for use as drug delivery device to prevent, or treat infections associated with orthopaedic implants.

We have proposed several target performance properties of the drug-loaded membranes in Table 1. Biocompatible for tissue integration Lifetime of nanofiber membrane!

Biodegradable to avoid surgical removal! Flexibility to conform to biological and implant surfaces Drug loading!

Loading of one or more antibiotic which shows efficacy against common Gram positive bacteria associated with orthopaedic infections Drug release!

Initial burst release to eradicate bacterial contamination at time of surgery! Controlled release above MIC over membrane lifetime to prevent hematological seeding of implant surface Other!

Chemical stability of drugs maintained 75 1. However, electrospinning is the most straight-forward, versatile, and cost-effective technique for the production of uniform, non-woven polymeric nanofibers Reneker and Chun, ; Frenot and Chronakis, ; Nair and Laurencin, B Blueprint drawing of the electrospinning apparatus highlighting the operational and solution conditions used in this thesis to form continuous, defect free PLGA nanofibers see section 4.

The electric field potential which deforms the fluid meniscus at the capillary tip, ejects the polymer solution into an axial stable jet, which accelerates and may splay repeatedly within the radiant electrical field.

As the fiber accelerates, instabilities force it to whip towards the ground collector as it stretches, thins, and solidifies into a uniform polymer fiber.

Fiber formation can be broken down into five distinct processes: 1 charging of the polymer fluid; 2 formation of the cone-jet; 3 thinning of the jet in the electrical field; 4 whipping instability and drying of the jet in the radiant electrical field; and 5 collection of the solidified fiber on a grounded target.

Charged ions, generated within the spin dope rapidly move in response to the charge, acting as a charge carrier to the bulk and in turn transfer a force vector to the bulk polymer solution pushing molecules and radiant charge towards the open capillary tip Reneker and Chun, As the field strength is increased, the spherical droplet begins to show signs of convexity as repulsion between like-charges at the free surface of the polymer solution work against surface tension.

At a critical electrical potential, slightly greater than the potential for conical equilibrium by overcoming surface tension, the distorted droplet becomes unstable and an axial jet is projected at the apex of the conical meniscus at the point of maximum field strength.

The initiation of the axial jet represents the beginning of fiber formation Taylor, ; Rutledge and Fridrikh, Upon ejection, the jet is accelerated and thins rapidly due to the viscoelastic properties of the spin dope.

The rapid thinning of the jet decreases bulk solution conductivity and results in a rapid increase in surface charge density.

Surface charge density increases until a near asymptotic value, at which point the jet thins more slowly as a result of only electrical potential Hohman et al.

In addition, as the distance between the jet lead and the spinneret increases, the electric potential of the jet decays exponentially.

At sufficient distance from the spinneret, electrostatic attraction forces oppose fluid inertial forces and cause the jet acceleration to slow down Ganan-Calvo, ; He et al.

Instabilities can result in jet splaying or splitting to form multiple jets. However, the most common instability associated with electrospun jets is bending or whipping instability.

The mechanism of whipping instability is strong charge repulsion at the surface of the thinning jet, where strong repulsion between neighboring charges forces an area of the jet to deviate perpendicular from the axial path.

Surface tension, inertia, and jet viscosity serve to counteract this instability by reducing surface area, velocity, and shear, respectively, resulting in relatively small perturbations that amplify as they travel down the jet path producing wave-like, or helix-like perturbations Figure 1.

These perturbations allow for further jet thinning and solidification into fibers Reneker et al. Electrified jets, and subsequent solidified fibers remain charged as they traverse the electrical field until they impact Taylor Cone Straight Jet Onset of First Cycle of Instability Onset of Second Cycle of Instability Onset of Third Cycle of Instability Collector 80 on a grounded, or lower potential electrode where they are collected.

The electrospinning setup is axial symmetric, and consequently, impact on the collector electrode with no preferred orientation results in a non-woven fiber membrane Rutledge and Fridrikh, Outside of these limiting conditions, weak charge repulsion within the hydrodynamic jet Lin et al.

Using their mathematical models of nanofiber formation, the authors divide operational and solution parameters into 3 categories based on their influence on the forming nanofiber.

Strongest effects come from jet radius, charge density, nozzle-to-collector distance, and viscosity, with moderate effects from polymer concentration, solvent vapor pressure, and electric potential, and minor effects from surface tension.

Accordingly, since the rate of diameter change governs the spinnability of a polymer solution, Rutledge suggests that there are a number of relevant fluid properties, including viscosity, density, surface tension, conductivity, and dielectric constant, and operational parameters, including flow rate, electric current, and applied field, that influence nanofiber diameter, and can be manipulated to alter the behavior of the electrospinning jet.

In each case, an increase in viscosity or density results in greater polymer chain interaction and entanglement, which play a major role in determining fiber morphology.

Shenoy et al. Using a modified approach, Gupta et al. Rg 3 2 Nav Equation 1. Normalizing the solution concentration to the critical concentration, Gupta et al.

According to Gupta, an increase in molecular weight, or polymer concentration, can significantly impact the critical concentration required for chain overlap Gupta et al.

Accordingly, similar analyses of entanglements have been evaluated using the Berry number. The surface tension and charge density in electrospinning are two other critical factors, and have been shown to significantly influence nanofiber morphology.

The longer the electrospun jet stays in a state of instability, the more the jet thins. Therefore, the addition of charging agents i. Similarly, Son et al.

Son et al. Yang et al. In both cases, these authors suggested that increasing the dielectric constant resulted in smaller nanofiber diameters due to higher solution charge density.

Yang and coworkers compared individual solvents with binary solvent mixtures, suggesting that nanofiber morphology is a property better controlled through small manipulations in binary solvent mixtures Yang et al.

Katti et al. The resulting nanofiber displays matrix-type morphology and the loading and subsequent release characteristics of any encapsulated drug will influenced by a number of variables previously discussed, including drug loading, drug-polymer compatibility, and polymer degradation rate see section 1.

The release mechanism of drugs from non-porous electrospun nanofibers is dominated by diffusional transport Xie and Buschle-Diller, The release of tetracycline and paclitaxel from polyester nanofibers was shown to follow Fickian diffusional release using swellable device models and when assuming release via a mono-dispersed cylinder system Xie and Wang, ; Xie and Buschle-Diller, However, if the nanofibers contain a nanoporous surface, a desorption-limited mechanism of release has been suggested, where interconnected nanopores serve to significantly increase the overall surface area of the nanofiber membrane Srikar et al.

Physicochemical properties of the encapsulated drugs can also play an important role in regulating drug release.

Drugs with favorable solubility in the polymer have been shown to disperse throughout the fiber forming a solid solution see section 1. Incompatible drugs, are found localized at the fiber surface, and display an uncontrolled burst phase of release Zeng et al.

Similarly, the amount of drug loaded into the nanofiber membrane can also influence drug release. An increase in drug loading has been associated with a greater burst phase of release Kim et al.

Although polymer properties, such as molecular weight Xie and Buschle-Diller, , can influence drug release, the degradation of nanofibers over the typical time courses studied, does not have a major influence on release rate.

In an interesting experiment, Puppi et al. Therefore, it was suggested that the influence of matrix erosion in ultra-fine nanofibers might not play an important role in drug release over moderate incubation times Xie and Buschle-Diller, Phase separation behavior of FA and RIF was examined using real- time video recordings of single microsphere formation via the solvent-evaporation method.

Phase diagrams were constructed illustrating the phase changes taking place from the liquid through to the final glassy states as microspheres form.

Accordingly, the following were the specific research objectives: 1 To characterize the solid state properties of FA Chapter 2.

However, a disproportionate increase in implant-associated infections has prevented the success of numerous restoration procedures.

The persistence of SSI following invasive surgery is due, at least in part, to the emergence of multi-drug resistant microorganisms. For instance, in Staphylococcus aureus alone, there are reports of resistance to "-lactams Ayliffe, , macrolides Schmitz et al.

In addition, clinical isolates from orthopaedic surgical sites have shown that methicillin-resistant S. Therefore, there is a clinical need for alternative treatment strategies.

Currently, there is much interest in the use of old generation antibiotics in surgical prophylaxis and treatment due to the decreased incidence of antibiotic resistance to these drugs Maviglia et al.

One class of antibiotics that has garnered attention is the steroidal antibiotics, specifically fusidic acid FA. FA is a tetracyclic triterpenoid derived from the fermentation broth of the fungus Fusidium coccineum Figure 1.

However, it has yet to be approved for use in the United States Howden and Grayson, FA has high antimicrobial activity against Gram- positive bacteria, in particular S.

It is due to this spectrum of activity that FA has become increasingly popular in the treatment of bone and prosthetic joint infections as Gram-positive organisms are commonly the culprits of orthopaedic infections.

Current guidelines state that FA is indicated for the treatment of prosthetic joints infections when MRSA is the infecting microorganism, and is dosed orally at mg every 8 h for a minimum of 3 months Zimmerli and Ochsner, ; Mastrokalos et al.

However, despite this dose being equivalent to intravenous dosing, maintaining minimum inhibitory concentrations at the site of infection as well as systemic side effects are often of concern.

Therefore, a primary focus of our lab is the development of a locally applied drug delivery system of FA for the treatment of orthopaedic infections.

The local administration of antibiotics to the site of infection is an attractive option for the treatment of bone infections due to the ability to maintain high tissue levels for prolonged periods of time, while simultaneously avoiding systemic side effects.

To date, several locally applied antibiotic delivery systems have been investigated including ciprofloxacin, gentamicin and vancomycin loaded poly methylmethacrylate bone cement beads Mader et al.

In many cases, these systems consist of solid drug particles dispersed throughout the device matrix, such as in the case of ciprofloxacin and fosfomycin loaded polyurethane films Schierholz et al.

Recently, our group has demonstrated that when FA is formulated in poly D,L-lactic acid-co-glycolic acid microspheres and films cast from dichloromethane DCM , the drug phase separates into drug- rich microdomains, suggesting that FA is present in the solid-state within the polymer Yang et al.

Even though FA has been in use for several decades and there is renewed interest in its use in implant associated infections, its solid-state properties have not been reported in the peer- reviewed literature.

The patent literature Jensen and Andersen, , and one monograph Reeves, , claim that FA free acid exists as a hemihydrate.

In this work, we describe the preparation and characterization of the commercially available solid, 2 new polymorphic forms, and an amorphous form of FA.

At predetermined time points, the suspension was filtered using a 0. All recrystallized forms of FA were stored under mmHg vacuum at ambient temperature until analysis.

The data were collected at a temperature of The crystal-to-detector distance was set to Samples were packed into a thin-walled capillary tubes special glass; Charles Supper Company, Natick, MA and sealed using a small amount of capillary wax.

Approximately 5 mg of sample was weighed into a hermetically sealed pan with a pinhole. The heat flow, and heat capacity of the instrument was calibrated using a high purity indium standard.

Samples were viewed using an Olympus CX41 transmission light microscope with magnification of 10x and a polarizing filter over the light source.

The thermal events were digitally recorded using Olympus Stream Basic imaging and documentation software v. A 50 mg sample of FA was compressed at psi for 60 sec in a 8mm electropolished die using an Enerpac P hydraulic press Butler, WI.

The die housing the drug compact was attached to a shaft and holder and submerged in mL degassed phosphate buffered saline PBS; pH 7.

Data were analyzed using Waters Millennium 32 software. All analysis was done in GraphPad Prism v. Based on single crystal XRD and thermal analysis findings, given below, we found no evidence of nydrate of solvate forms of FA.

Major diffraction peaks were present at 7. Amorphous FA lacked any diffraction peaks and was characterized by a characteristic broad halo.

The identification of the various polymorphs with respect to their source, and crystallization conditions are summarized in Table 2.

Form IV Tm,2 Hf Hf , whereas exothermic transitions are expressed as the heat of recrystallization!

To investigate the packing orientation of FA in this polymorph, a crystal with approximate dimensions of 0. Figure 3 shows the crystal structure of FA, highlighting the oxygen atoms O1-O6 involved in inter-molecular hydrogen bonding between FA molecules in the unit cell.

The unit cell of Form I is a primitive monoclinic lattice system containing two FA molecules Figure 2. Form III, either the commercially available form or through H2O-mediated polymorphic transformation of Form IV or amorphous FA, was composed of very small plates, with some scattered irregular particles.

Amorphous FA was a mixture of particles, with no distinct crystal habit. Thermal degradation events observed by TGA are summarized in Table 2. The thermal events observed by DSC are summarized in Table 2.

Polarized light was used to distinguish crystalline material, which appeared as bright birefringent areas, from non-crystalline material, which appeared as dark grey regions.

The amorphous form of FA was devoid of birefringence throughout the entire heating cycle. During solvent-mediated polymorphic transition, the solution is saturated with respect to the metastable polymorph, and supersaturated with respect to the most stable polymorphic form.

Thus, with time, the most stable form will nucleate and precipitate out to maintain thermodynamic equilibrium Miller et al.

Recent reports highlight the importance of proper solvent selection in screening for stable polymorphic forms Gu et al. These authors suggest that the hydrogen-bonding propensity HBP of the solvent and the overall solubility of the drug are the critical determinants for producing the most stable polymorph.

Solvents with high HBP but very poor drug solubility will retard polymorphic transformation favoring a metastable form. Conversely, solvents with weak HBP and higher solubility will favor a more stable polymorph Gu et al.

Single crystal XRD analysis of Form I yielded a primitive monoclinic lattice system containing two FA molecules, with no evidence of the presence of H2O or solvent within the lattice Figure 2.

We were unable to generate crystals large enough for single crystal XRD analysis from the other polymorphic forms, and therefore it is unclear how the molecular packing arrangements may differ between the solid forms.

Additionally, there is no reference pattern published in peer-reviewed literature, or in any online database i. Therefore, this report represents the first published single crystal XRD analysis of FA in the peer-reviewed literature.

Heating Form IV resulted in several thermal events. However, due to thermal degradation, as evidenced by sample discoloration and degradation peaks upon HPLC analysis, we were unable to characterize this mixture further.

The patent literature claims that FA exists as a hemihydrate Jensen and Andersen, , which would be characterized as weight loss in a stepwise fashion in a TGA analysis.

This was not observed in our experiments, and we found no evidence that the polymorphic forms of FA investigated in this work were hydrates.

This weight loss was attributed to degradation and vaporization of the samples and was further supported by the discolored appearance of the samples upon completion of the heating cycle.

Given the pKa of 5. However, the concentration of FA in the dissolution media at pH 3 was below the level of detection, and therefore we were unable to quantify the IDR.

Thus, for comparison between all forms, the IDR was evaluated at a physiologically relevant pH of 7. It is well known that polymorphism can have a dramatic impact on the solubility of a drug, with more stable polymorphs leading to lower dissolution rates as compared to their metastable counterparts Grant, This phenomenon is supported by a literature survey of 55 polymorphic compounds by Pudipeddi and Serajuddin who demonstrated that the vast majority of polymorphic forms exhibit limited spread in solubility ratios solubility of any polymorphic form, normalized to the solubility of the most stable form or dissolution rates, and is likely due to undetectable differences in the entropy between polymorphs Pudipeddi and Serajuddin, Our experiments confirm the existence of Form I and III, which have been previously reported in the patent literature.

Differences in dissolution rates between the polymorphs could conceivably have an impact on the release rate of FA from local delivery devices if the drug was present in the solid-state as Form IV or amorphous FA.

However, it is cautioned that formulation of FA as one of theses forms may lead to interconversion to Form III in the presence of an aqueous milieu potentially resulting in a reduction in the release rate of the drug.

Drug loaded microspheres injected subcutaneously, intramuscularly or locally at target sites can provide controlled release of bioactive agents over days, weeks or months to treat a variety of disease states Sinha and Trehan, ; Menei et al.

Accordingly, there are several successful marketed PLGA microsphere formulations loaded with drugs, such as leuprolide acetate, triptorelin pamoate, octreotide acetate, lanreotide, risperidone, naltrexone and exenatide, for the treatment of a range of conditions, including alcohol dependence, prostate cancer, acromegaly, endometriosis, and type II diabetes Letchford et al.

Recently, our group examined the loading of fusidic acid FA , an antimicrobial agent active against a number of Gram positive microorganisms including S.

In these studies, very distinctive microsphere morphology was observed as a result of phase separation of FA from PLGA as FA-rich, amorphous microdomains producing uniform and spherical protrusions on the microsphere 2 The data presented in the chapter have been published.

Gilchrist S. Upon formation of the initial oil in water emulsion, DCM rapidly leaves the dispersed phase, diffusing into the surrounding aqueous, continuous phase.

Consequently, the polymer and FA solution concentrates, and just before solidification of the polymer, a FA-rich DCM phase appears to separate.

Using a variety of techniques, including real-time recording of single-microsphere formation, it was shown that coalescence of these liquid FA-rich microdroplets throughout the forming microsphere and at the interface, resulted in the appearance and growth of rounded protrusions at the surface Yang et al.

This same phenomenon has also been observed by two other groups studying the encapsulation of cyclosporine A in PLGA microspheres Passerini and Craig, ; Malaekeh-Nikouei et al.

We agree with this conclusion, and our single microsphere studies directly demonstrated a similar phase separation process for FA Yang et al.

Phase separation of drug within the polymer matrix and at the surface of microspheres can occur during fabrication, most commonly using the solvent evaporation method, and has been extensively reported O'Donnell and McGinity, ; Li et al.

It is well established that drug may precipitate in the polymer in a crystalline form or as an amorphous form within the carrier Bodmeier and McGinity, ; Rosilio et al.

Interestingly, phase separation may be accompanied by changes in microsphere morphology from the completely smooth surfaces observed for control no drug microspheres to a concave dimpled appearance, as in, for example, paclitaxel loaded poly L-lactic acid microspheres Liggins and Burt, and progesterone loaded PLGA microspheres Rosilio et al.

Phase separation of drug in a number of binary and ternary solvent- cast films has also been reported Lu and Zografi, ; Panyam et al. Several reports have shown surface-associated drug phase separation from the polymer matrix, after the microspheres have been formed.

However, there are only a few reports attempting to explain the underlying mechanisms of formation behind these phenomena. DeLuca and co-workers have carried out detailed kinetic and thermodynamic modeling of the formation of polymeric microspheres using the solvent evaporation method, and the processes underlying the formation of porous microspheres Li et al.

In these investigations, porous microspheres were formed when salmon calcitonin peptide was loaded into PLGA microspheres using dichloromethane DCM as solvent and methanol as a cosolvent to dissolve the loaded peptide.

The authors explain that as solvent is transported out of the liquid phase droplet, the liquid droplet becomes increasingly viscous and eventually forms a gel at what is termed the viscous boundary VB.

Loss of more solvent results in crossing the glassy boundary, eventually leading to a glassy state composed of solid polymer and drug Li et al.

Li et al. Using the micropipette techniques discussed in this investigation, it is possible to view, in real time, the events of phase separation to confirm or refute any of these postulated mechanisms.

An understanding of the phase behavior of drugs, and methods to study these phenomena in polymeric carriers are critical for the optimization of drug-delivery formulations, as phase separation can impact drug release profiles and drug stability.

RIF is active against S. Therefore, a combination product that provides controlled release of FA and RIF at the site of infection may be of clinical benefit.

In this work, we explore the ternary phase separation behavior and miscibility characteristics of FA and RIF in PLGA microspheres prepared by solvent evaporation from DCM, and bring a new mechanistic understanding to these complex, and thus far unseen, phase separation behaviors using real-time video recording of forming microspheres.

The resulting emulsion was stirred continuously for 2. The microspheres were allowed to sediment in the flask on the bench top for 5 min and subsequently washed three times with distilled water with bench top sedimentation for 5 minutes to remove unencapsulated drug, residual DCM and PVA.

The microspheres were then dried under mmHg vacuum at room temperature for 24 h using a bench top vacuum oven Napco No.

The particle size distribution of microspheres was expressed as mean diameter along with the span, where span is calculated according to the following equation: Equation 3.

Ascorbic acid was added to the PBS release media as an antioxidant, since it has been shown to stabilize RIF and prevent oxidative degradation Weber et al.

The remaining media was removed and replaced with fresh PBS pH 7. The solubility of FA is 5. The heat flow and heat capacity of the instrument was calibrated routinely using a high purity indium standard.

The Fox equation is expressed as: Equation 3. Rickard et al. The droplet was observed and the image digitally recorded as the DCM diffused into the surrounding aqueous phase, resulting in a solidified microsphere.

The experimental setup includes an inverted optical microscope Diaphot ; Nikon, Melville, NY, USA with a 40" or an oil-immersion " objective lens, micropipette manipulation system, and video capturing equipment.

The chamber used with the 40" objective lens was a standard glass cuvette 2-mm path length Duncan and Needham, ; Rickard et al.

For experiments with the oil-immersion objective, the chamber was formed from coverslips 22 " 30 mm and cut glass microscope slides Duncan and Needham, Coverslips were affixed to the top and bottom of the glass slide pieces with vacuum grease to create a chamber with final dimensions of approximately 22 " 25 " 2 mm, which was open on two sides.

The initial solution density,! The solute concentration was then calculated as a function of time based on the volume change of the droplet according to!

The solute concentration at the time of phase separation was calculated according to the diameter of the droplet when the first signs of phase separation were visible as indicated in Figure 3.

The initial mass is given by!!! The interstitial surface space between these spherical protrusions showed a dimpled appearance characteristic of pure RIF.

Detailed surface morphologies for both initial and drug-released released microspheres are shown in Figure 3.

DCM Figure 3. FA-loaded microspheres showed an increase in mean diameter with the co-loading of RIF in a dose dependent manner with mean diameters of In contrast, RIF-loaded microspheres demonstrated a decrease in mean diameter with FA co-loading, with mean diameters of The physical characteristics of the microspheres are summarized in Table 3.

The peak temperature of the first endothermic transition in the first heating cycle. Integration of the first endothermic transition in the first heating cycle.

The midpoint of the heat capacity change in the first heating cycle. Measured by the span, according to Equation 3. A smaller span indicates a narrow size distribution.

Recovered drug expressed as a percentage of the theoretical drug loading. Unfortunately, the high scattering intensity of RIF precluded the ability to map the microsphere surface for actual drug localization; however, we were able to map the surface of the co-loaded microsphere for PLGA distribution.

As expected from a reciprocal combination of the mixtures, the measured Tg for the first heating cycle was dependent on the FA and RIF content of the co- loaded microspheres.

The Tg for co-solidified drug and microspheres is illustrated in Figure 3. Note that, in order to see the details of microstructure and phase separations, a higher magnification oil immersion lens was used to obtain the images in Figure 3.

This necessitated studying smaller microspheres than those that provided the data in Figure 3. In the absence of FA, there are no water inclusions, but the aqueous solubility of FA is 5.

With further loss of DCM solvent and shrinking of the microdroplet volume, these FA-rich microdroplets within the forming microsphere coalesced to form larger microdroplets.

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