This summary provides an overview of the key points from the document: 1) The document describes a project to develop a nanoparticle system to increase chronic wound healing rates by targeting impaired mechanisms in diabetic and elderly patients. 2) Current wound healing solutions are expensive and not optimal, with the average time to fully close a wound being over 5 months. 3) The proposed concept utilizes nanoparticles to release growth factors into wounds over days to weeks in order to promote healing and reduce needed applications. 4) Mathematical modeling of growth factor diffusion and release kinetics from nanoparticles was performed to prove feasibility, and nanoparticles were successfully synthesized with desired properties.

1. Yunqi He, Anthony Yung, Thomas Chedid, Abhinav Damaraju
Senior Design Final Report
Advisor: Dr. François Berthiaume
Nanoparticles for Improved Skin Wound Healing
Introduction
Chronic wounds, occurring mainly in elderly and diabetic individuals, represent a
significant economic and health care burden in the US. Current wound healing solutions are
expensive and do not target the impaired wound healing mechanisms of diabetic and elderly
patients. As a result, optimal healing rates are not achieved. The goal of the project is to develop
a nanoparticle system that can increase chronic wound healing rates by addressing some of the
underlying causes of the inhibited wound healing response of diabetic and elderly patients.
Background
There are currently an estimated 6.5 million people suffering from chronic wounds in the
US. Each wound costs $10,376 on average to heal, making the wound healing solution market
huge at approximately $67.4 billion spent annually [1]. Chronic wounds, characterized as
painful, unappealing, and prone to infection, often render patients immobile and result in extreme
inconvenience. Because chronic wounds arise from exacerbated wound healing processes such
as poor vascularization and cell migration, these wounds are very hard to heal. In fact, the
average time it takes to fully close a wound is 165 days, or a little over 5 months [1]. For more
complicated wounds such as diabetic foot ulcers, where there is an increased inflammatory
response, decreased amounts of growth factors, and elevated protease concentration, there is
even a chance that the wound won’t heal with treatment, often resulting in chronic infections and
amputations. According to the National Institute of Diabetes and Digestive and Kidney
Diseases, 40% of the people with chronic wounds have diabetic foot ulcers and around 24% of
these people or 6 million need amputations [2].
There are numerous technologies on the market to treat chronic wounds although no single
treatment is optimal. For instance, one product on the market right now is vacuum assisted
closure devices, which use negative pressure to suction wounds close. The advantage of these
devices is that it provides a sealed wound environment that minimizes bacterial infection.
However, these devices are expensive and greatly reduce patient’s mobility. Next, there are skin
substitute products. These mimic the extracellular matrix of skin and provide a network for the
cell proliferation [3]. Nevertheless, research has shown that skin substitutes are at most only
about 50% effective in completely healing chronic wounds [4]. To target the issue of reduced
amounts of growth factors in chronic wounds, some companies have developed platelet-derived
growth factors that can be applied topically onto the wound. This method is still not very
effective because the high concentration of proteases in the diabetic wound environment cleave
growth factors upon entering without protection. Proteolytic degradation of growth factors is
one of the main reasons for the unsuccessful healing of chronic wounds so targeting this
mechanism is crucial. In addition, long term use of these platelet-derived growth factors will
have a carcinogenic effect. Lastly, some companies have tried to use mesenchymal stem cells
(MSCs) to promote healing. MSCs promote angiogenesis and decreases tissue inflammation but
the disadvantage is that they don’t adhere to wounds well. Clearly no current solution effectively
solves the chronic wounds problem even though the market has many products aiming to treat
wounds. As a result, there is a high demand in creating an active and efficient wound healing
solution to target chronic wounds.
Concept Design and Engineering Constraints

2. In order to improve current wound healing solutions, there are several key criteria that we
must consider. First, the reduction in healing time is by far the most important key criterion that
we must fulfill. On average, current skin substitute products heal wounds completely in 5 weeks.
Our target specification is to heal completely in 3 weeks. Second, the solution must be cost
effective. The cost of resources needed to develop the solution should be relatively low and
cannot be higher than the possible revenue so there is a potential for earning a profit. Third, we
should minimize the side effects and safety concerns. Any therapeutic adverse effect will
disregard our effort and purpose of wound healing. The fourth criterion is that the solution must
have a convenient application. This will save cost by avoiding complicated surgery and reducing
time of hospital stay. A convenient application will require less maintenance and necessary
reapplications. The ideal target is that our solution would be applied once a week at most. Aside
from the above requirements, it is in our best interest to have a product similar to the current
standard of care because medical practitioners usually don’t want to change their methods of
treatment drastically. Our solution should aim to fit into the current standard of care, so it will be
easier for doctors to adopt. Because key criteria are crucial for the success of our solution, all key
specifications will be addressed in our proposed concept.
Our proposed concept is the utilization of nanoparticles to release growth factors into the
wound. Nanoparticles will be produced by (a) linking charged peptides to phospholipids through
electrostatic attraction and (b) binding growth factors to peptides. The configuration of
nanoparticles protects growth factors from accelerated degradation in vivo due to proteases and
allows for controlled release of growth factors over a period of days to weeks. The growth
factors ultimately remain longer in the wound area and penetrate deeper into the tissue, resulting
in the growth of new tissue. Overall, this process should allow a higher rate of healing and
therefore reduce the amount of applications need to close the wound. We will minimize risk and
simplify regulatory concerns by using FDA approved growth factors. Our nanoparticles can be
applied as a cream or incorporated into existing wound healing products. This convenient
application will surely be easy for doctors to use.
Materials and Methods
To proof the feasibility of our concept, we first used MATLAB to mathematically model
the release of growth factors from nanoparticles into the wound. The nanoparticle system was
represented as diffusion of a solute from a slab as shown in Figure 1, where the solute
corresponds to nanoparticles and the slab consists of a porous material (e.g. skin substitute) that
is applied onto the wounded tissue.
Figure 1: The nanoparticles are uniformly distributed within microporous slab of radius R and
thickness L. Since R>>>L, all transport is effectively along the thickness of the material and it is
as if the slab edge was coated with an impermeable material. Therefore, all solute exits the slab
to reach the wound surface.

3. The equation used in the model was Fick’s second law of diffusion with the following set of
boundary conditions: (1) t=0, C(x,t)=C0, (2) x=0, dC/dx = 0, and (3) x= L, C=0. C is the
concentration of the growth factors (mM), D is the diffusivity of the growth factors in wounded
tissue (cm2/s), x is the distance away from the surface (m) and t is time (s). We have set the
initial concentration C0 = 10-6 mM and D = 10-3 cm2/s. A source term was then incorporated into
our model to account for the release of the growth factor from the nanoparticles:
After modeling the behavior of the nanoparticles, we move on to the synthesis of
nanoparticles. Components of the nanoparticles were selected after consideration of size, charge,
and ability to monitor cellular response. To form the liposome core of the nanoparticles, we used
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-distearoyl-sn-glycero-
3-phosphatidic acid (DSPA) at a molar ratio of 7:2:1. The liposomes were also labeled with 1%
rhodamine for fluorescence analysis. The liposomes were extruded through 200 nm and 100 nm
polycarbonate membranes to acquire the necessary size and finally deca-lysine was mixed with
the liposomes. Deca-lysine was used here because it has a positive charge and therefore
associates with negatively charged liposomes; furthermore, deca-lysine can be linked to growth
factors using simple conjugation methods. The sizes of the generated nanoparticles were
analyzed by dynamic light scattering (DLS). To assess whether the peptides attached to the
liposome, size exclusion chromatography followed by fluorescence assays were performed.
Results and Discussion
Modeling results: After deriving Fick’s second law using the predetermined boundary condition,
analytical solution that represents the release of growth factor was obtained as following:
The analytical solution of the release of growth factor
was then computed into MATLAB and a concentration profile of the release of growth factor
was obtained (Figure 2). The trend on Figure 2 confirmed that as time and distance increases the
concentration of growth factor decreases, signifying growth factor is penetrating into the wound.
An arbitrary source term was then added into the model to represent the release of growth factor
from nanoparticles (Figure 3). What we can interpret from Figure 3 by the increase in
concentration beyond the initial concentration (straight blue line) is that there is a higher chance
of growth factor molecules entering the wound as more growth factor is released although the
similar pattern of decreased concentration with increase in time and distance exists as shown in
Figure 2. Finally, three separate half-life cases for the release kinetics of growth factor from
nanoparticles, 1 hour, 1 day, and 1 week, were evaluated on the concentration profile (Figure 4).
It was found that nanoparticles with a short half-life of 1 hour (Figure 4A) will mostly be
degraded before a significant amount of growth factor has had time to reach the wound; in effect
this would be equivalent to putting the free growth factor in a soluble form into the slab.
Nanoparticles with a longer half-life of 1 day (Figure 4B) and 1 week (Figure 4C) would see
slower and more sustained release of growth factor towards the wound due to a slower
degradation rate. Since ideally a sustained delivery of growth factor towards the wound is more
desirable, our nanoparticles should aim to have a half-live as long as possible, preferably close to
1 week.

4. Figure 2: Concentration profile of nanoparticles. Figure 3: Concentration profile with
Each line represents different time points. source term at various time points.
Figure 4: Concentration profile of nanoparticles with three different half-lives: (A) half-life of 1
hour, (B) half-life of 1 Day, and (C) half-life of 1 Week
Experimental results: Measurements from the DLS show that the size of the liposomes without
the peptide was around 140 nm in size, which is over two orders of magnitude less than the pore
size of FDA-approved skin substitutes. This signifies that incorporating nanoparticles into
existing skin treatments is possible. However, the average size of the liposomes with the peptide
attached was greater than 1000 nm, suggesting a possibility of liposome aggregation. Data from
size exclusion chromatography (Figure 7) similarly showed that there is aggregation of the
material due to the large (green) peak between fraction number 1 and 2. Nonetheless, the main
rhodamine fluorescence elution peak (associated with liposomal nanoparticles) for the
phospholipids alone, shown at fraction number 3, was slightly delayed compared to
phospholipids with the peptide, shown at fraction number 2, thus indicating only a slightly
greater size for the former. The relatively similar position of the two peaks also suggested that
the peptide indeed attached to the liposomes, forming nanoparticles. This will need to be
confirmed using other methods, such as zeta-potential measurement. Lastly, there is a smaller
red peak at around fraction numbers 8 to 10, indicating that some peptides did not bind to the
liposomes.

5. Figure 7: Elution profiles of liposomes with peptides and empty liposomes, obtained by size
exclusion chromatography
Conclusion
The completion of the MATLAB model allowed us to have a good understanding of the
general behavior of our nanoparticles and how certain factors such as regeneration rate and
degradation rate of nanoparticles affect this behavior. With this understanding, we can tailor
these factors to reach optimal healing results of our nanoparticles. Furthermore, data from DLS
and size exclusion chromatography revealed that the synthesis of nanoparticles has been
successful although there might be aggregation of liposomes and considerable amounts of
unbound peptide. Future works would therefore include first finding methods to limit
aggregation and maximized the amount of peptides bound to nanoparticles, such as PEGylation
of lipids, and then the attaching of growth factors onto the nanoparticles followed by in vitro
studies of protease cleavage and cellular responses.
Reference
1. Sen CK, Gordillo GM, Roy S, Kirsner R, Lambert L, Hunt TK, Gottrup F, Gurtner GC,
Longaker MT. Human skin wounds: a major and snowballing threat to public health and the
economy. Wound Repair Regen. 2009; 17:763–71.
2. "Diabetic Ulcers." Diabetic Ulcers. N.p., n.d. Web. 03 Dec. 2013.
3. Halim, Ahmad Sukari, Teng Lye Khoo, and Shah Jumaat Mohd. Yussof. "Abstract."
National Center for Biotechnology Information. U.S. National Library of Medicine, 01 Mar.
0006. Web. 04 Dec. 2013.
4. Przyborowski, Melissa. "Strategies for Improving Growth Factor Function in Diabetic
Wounds." Rutgers University Biomedical Engineering Building, Piscataway. July 18, 2012.
Thesis Proposal Defense.