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A phage-targeting strategy for the design of spatiotemporal drug delivery from grafted matrices.

Sawada R, Peterson CY, Gonzalez AM, Potenza BM, Mueller B, Coimbra R, Eliceiri BP, Baird A - Fibrogenesis Tissue Repair (2011)

Bottom Line: After three to six rounds of biopanning, phage recovery and phage amplification of the bound particles, any phage that had acquired a capacity to bind the matrix was sequenced.In this first report, we identify distinct classes of matrix-binding peptides which elute differently from the screened matrix and demonstrate that they can be applied in a spatially relevant manner.We suggest that further applications of these combinatorial techniques to wound-healing matrices may offer a new way to improve the performance of clinically approved matrices so as to introduce temporal and spatial control over drug delivery.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, Division of Trauma, Surgical Critical Care and Burns, University of California San Diego School of Medicine, 200 W, Arbor Dr,, San Diego, CA 92103-8236 USA. anbaird@ucsd.edu.

ABSTRACT

Background: The natural response to injury is dynamic and normally consists of complex temporal and spatial cellular changes in gene expression, which, when acting in synchrony, result in patent tissue repair and, in some instances, regeneration. However, current therapeutic regiments are static and most rely on matrices, gels and engineered skin tissue. Accordingly, there is a need to design next-generation grafting materials to enable biotherapeutic spatiotemporal targeting from clinically approved matrices. To this end, rather then focus on developing completely new grafting materials, we investigated whether phage display could be deployed onto clinically approved synthetic grafts to identify peptide motifs capable of linking pharmaceutical drugs with differential affinities and eventually, control drug delivery from matrices over both space and time.

Methods: To test this hypothesis, we biopanned combinatorial peptide libraries onto different formulations of a wound-healing matrix (Integra®) and eluted the bound peptides with 1) high salt, 2) collagen and glycosaminoglycan or 3) low pH. After three to six rounds of biopanning, phage recovery and phage amplification of the bound particles, any phage that had acquired a capacity to bind the matrix was sequenced.

Results: In this first report, we identify distinct classes of matrix-binding peptides which elute differently from the screened matrix and demonstrate that they can be applied in a spatially relevant manner.

Conclusions: We suggest that further applications of these combinatorial techniques to wound-healing matrices may offer a new way to improve the performance of clinically approved matrices so as to introduce temporal and spatial control over drug delivery.

No MeSH data available.


Related in: MedlinePlus

Peptide screening, dose-response analyses and spatial immobilization of targeted particles onto intact matrices. Using the methods described in the text, individual phage were tested for either their ability to bind to immobilized components (for example, chondroitin sulfate, collagen 4, bovine serum albumin or to sonicated matrix samples, as illustrated by (A) binding of IT8 peptides to immobilized chondroitin sulfate or (B) dose-response analyses to specific peptides binding to sonicated matrix samples immobilized in ELISA wells. Spatial binding to matrix sections was established by placing targeted particles as (C) continuous lines along two sides (sample 2) or as a dot (sample 3). Phosphate-buffered saline alone was negative (sample 1). In all instances, binding of particles was assessed by chemiluminescence.
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Figure 8: Peptide screening, dose-response analyses and spatial immobilization of targeted particles onto intact matrices. Using the methods described in the text, individual phage were tested for either their ability to bind to immobilized components (for example, chondroitin sulfate, collagen 4, bovine serum albumin or to sonicated matrix samples, as illustrated by (A) binding of IT8 peptides to immobilized chondroitin sulfate or (B) dose-response analyses to specific peptides binding to sonicated matrix samples immobilized in ELISA wells. Spatial binding to matrix sections was established by placing targeted particles as (C) continuous lines along two sides (sample 2) or as a dot (sample 3). Phosphate-buffered saline alone was negative (sample 1). In all instances, binding of particles was assessed by chemiluminescence.

Mentions: The strategy to confer temporal and spatial control to drug release on the matrices involves the use of peptides with differential affinities to components of the deployed graft. To this end, we evaluated peptide binding to the matrix relative to controls by direct incubation onto the target. Representative results for different targeting peptides are presented in Figure 8. The initial screen involved direct binding analyses of the targeted particles as illustrated for the IT8 peptides recovered in the presence or absence of blocking BSA (Figure 8A). Under the ELISA conditions used (see Methods), background binding was low and there was an approximately 105 increase in binding. Although all of the peptides selected as candidates showed specific binding, some (for example, IT8-R4PA21) seemed to have greater capacity. This affinity is evaluated in dose response curves which, showed a dose -dependent increase in binding that reflects the affinity the peptides confer to the particles (Figure 8B). In this specific example, binding of peptides selected by chondroitin sulfate displacement was evaluated in an ELISA using the glycosaminoglycan to capture targeted phage. Although there was a 105-fold increase in saturable binding, the affinities were narrow and detectable over a two- to three-fold change in concentrations. In-depth analysis of specificity, capacity, affinity and avidity of all the peptide candidates is underway on matrix, sonicated samples and GAG-coated plates to select targeting leads for evaluation in preclinical studies.


A phage-targeting strategy for the design of spatiotemporal drug delivery from grafted matrices.

Sawada R, Peterson CY, Gonzalez AM, Potenza BM, Mueller B, Coimbra R, Eliceiri BP, Baird A - Fibrogenesis Tissue Repair (2011)

Peptide screening, dose-response analyses and spatial immobilization of targeted particles onto intact matrices. Using the methods described in the text, individual phage were tested for either their ability to bind to immobilized components (for example, chondroitin sulfate, collagen 4, bovine serum albumin or to sonicated matrix samples, as illustrated by (A) binding of IT8 peptides to immobilized chondroitin sulfate or (B) dose-response analyses to specific peptides binding to sonicated matrix samples immobilized in ELISA wells. Spatial binding to matrix sections was established by placing targeted particles as (C) continuous lines along two sides (sample 2) or as a dot (sample 3). Phosphate-buffered saline alone was negative (sample 1). In all instances, binding of particles was assessed by chemiluminescence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3050739&req=5

Figure 8: Peptide screening, dose-response analyses and spatial immobilization of targeted particles onto intact matrices. Using the methods described in the text, individual phage were tested for either their ability to bind to immobilized components (for example, chondroitin sulfate, collagen 4, bovine serum albumin or to sonicated matrix samples, as illustrated by (A) binding of IT8 peptides to immobilized chondroitin sulfate or (B) dose-response analyses to specific peptides binding to sonicated matrix samples immobilized in ELISA wells. Spatial binding to matrix sections was established by placing targeted particles as (C) continuous lines along two sides (sample 2) or as a dot (sample 3). Phosphate-buffered saline alone was negative (sample 1). In all instances, binding of particles was assessed by chemiluminescence.
Mentions: The strategy to confer temporal and spatial control to drug release on the matrices involves the use of peptides with differential affinities to components of the deployed graft. To this end, we evaluated peptide binding to the matrix relative to controls by direct incubation onto the target. Representative results for different targeting peptides are presented in Figure 8. The initial screen involved direct binding analyses of the targeted particles as illustrated for the IT8 peptides recovered in the presence or absence of blocking BSA (Figure 8A). Under the ELISA conditions used (see Methods), background binding was low and there was an approximately 105 increase in binding. Although all of the peptides selected as candidates showed specific binding, some (for example, IT8-R4PA21) seemed to have greater capacity. This affinity is evaluated in dose response curves which, showed a dose -dependent increase in binding that reflects the affinity the peptides confer to the particles (Figure 8B). In this specific example, binding of peptides selected by chondroitin sulfate displacement was evaluated in an ELISA using the glycosaminoglycan to capture targeted phage. Although there was a 105-fold increase in saturable binding, the affinities were narrow and detectable over a two- to three-fold change in concentrations. In-depth analysis of specificity, capacity, affinity and avidity of all the peptide candidates is underway on matrix, sonicated samples and GAG-coated plates to select targeting leads for evaluation in preclinical studies.

Bottom Line: After three to six rounds of biopanning, phage recovery and phage amplification of the bound particles, any phage that had acquired a capacity to bind the matrix was sequenced.In this first report, we identify distinct classes of matrix-binding peptides which elute differently from the screened matrix and demonstrate that they can be applied in a spatially relevant manner.We suggest that further applications of these combinatorial techniques to wound-healing matrices may offer a new way to improve the performance of clinically approved matrices so as to introduce temporal and spatial control over drug delivery.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, Division of Trauma, Surgical Critical Care and Burns, University of California San Diego School of Medicine, 200 W, Arbor Dr,, San Diego, CA 92103-8236 USA. anbaird@ucsd.edu.

ABSTRACT

Background: The natural response to injury is dynamic and normally consists of complex temporal and spatial cellular changes in gene expression, which, when acting in synchrony, result in patent tissue repair and, in some instances, regeneration. However, current therapeutic regiments are static and most rely on matrices, gels and engineered skin tissue. Accordingly, there is a need to design next-generation grafting materials to enable biotherapeutic spatiotemporal targeting from clinically approved matrices. To this end, rather then focus on developing completely new grafting materials, we investigated whether phage display could be deployed onto clinically approved synthetic grafts to identify peptide motifs capable of linking pharmaceutical drugs with differential affinities and eventually, control drug delivery from matrices over both space and time.

Methods: To test this hypothesis, we biopanned combinatorial peptide libraries onto different formulations of a wound-healing matrix (Integra®) and eluted the bound peptides with 1) high salt, 2) collagen and glycosaminoglycan or 3) low pH. After three to six rounds of biopanning, phage recovery and phage amplification of the bound particles, any phage that had acquired a capacity to bind the matrix was sequenced.

Results: In this first report, we identify distinct classes of matrix-binding peptides which elute differently from the screened matrix and demonstrate that they can be applied in a spatially relevant manner.

Conclusions: We suggest that further applications of these combinatorial techniques to wound-healing matrices may offer a new way to improve the performance of clinically approved matrices so as to introduce temporal and spatial control over drug delivery.

No MeSH data available.


Related in: MedlinePlus