Aggregation and Dendrimer Mediated Secondary Binding towards Folate Binding
Protein &Fatigue Failure Mechanism of Anterior Cruciate Ligament FracturebyJunjie Chen

A dissertation submitted in partial fulfillmentof the requirements for the degree ofDoctor of Philosophy(Chemistry)in the University of Michigan
2018

Doctoral Committee:
Prof. Mark Banaszak Holl, co-chair
Prof. Neil Marsh, co-chair
Prof. Bradford OrrProf. Ayyalusamy Ramamoorthy

Junjie Chen
junjchen@Umich.edu
ORCID iD: 0000-0001-8004-8159

DEDICATION
For my family, my wife and my advisorsiiAcknowledgements
This work won’t be possible without the help from my advisor, Dr. Mark Banaszak
Holl. Mark is always accessible to his students to talk about science and even other life
advices. As one of the greatest mentors at the University of Michigan, Mark always keeps
his door open for students whenever he is available. Mark forwarded many great
opportunities including paper review, paper invitation and internships, all of which
benefitted my career. Mark is very patient on my growth and tolerated the tons of stupid
mistakes/accidents I made in the past five years. When I was a GSI but got stuck in China
due to delayed visa in winter 2016, Mark even taught for me for one week. I also learned
the “smell of science” in the Banaszak Holl lab – how to set up scientific a question toanswer; how to support your hypothesis with the “one-million control” and how to translate
your result to the science community. Michigan lost a great professor and I feel that
Monash University is extremely lucky to have Mark joining their team in 2018.
The Banaszak Holl group members have created a fun and collaborative
atmosphere throughout my PhD. Rachel Merzel, who entered the group in the same yearwith me, had a deep collaboration on countless experiments. To date, we have publishedfour papers together, ranging from dendrimer conjugation to protein aggregation, and toanimal test on drug delivery. Rachel leaves a significant impact on at least half of my PhD.

iii

I also need to thank Dr. Casey Dougherty and Dr. Sriram Vaidyanathan for providing thehelps and suggestions early in my PhD. Jinhee Kim is my major collaborator on chapter 3
and she helped establish the understanding of ACL damage. I am also grateful for Isabel
Colon-Bernal and Ted Ahn for maintaining the lab environment and equipment.
I can’t be more grateful for my other committee members and my collaborators. Dr.
Orr, who attends group meetings throughout my PhD, gave numerous helpful suggestions
on polishing my work and leading to publications. Dr. Marsh provided extensive support
in all the protein related material in this thesis as well as the collaboration opportunity on
protein cages with Ajitha Cristie-David. Dr. Ramamoorthy provided NMR support for
many projects and the opportunity to work on lipid nano-disks. I also initiated manycollaborations with many talented people on campus. It was my hornor to publish with Dr.
Zhan Chen, Minyu Xiao, Dr. Wojtys, Dr. Schlecht, Dr. Ashton Miller, and Haiye Wang etal. I learned additional skills/knowledge from those collaboration and those fun experience
are one of the most rewarding things I got in graduate school.
The staff and researchers at University of Michigan provided all the equipment andsupport to make everything possible. The Department of Chemistry staff created a safe andconvenient working environment for my study. The Michigan Nanotechnology Instituteiv

for Medicine and Biological Sciences (MNIMBS) provided many technical support for the
instrument and special thanks to Dr. Pamela Wong.
My parents have been very supportive both mentally and financially of me studying
abroad. I felt deeply sorry for only visiting home once in my past four and half years. I
wish I could spend more time with them and give my full support back to them in the future.
Youfei, my fiancé, offered some of the happiest moments as I forged through the final
years of my PhD. I am looking forward to forming the future family with Youfei andpotentially, a collaborative scientific paper together.

v

Table of Contents
Dedicationii

Acknowledgementsiii

List of Figuresvii

Abstract

viii

Chapter
I. Enhanced Folate Binding Protein Avidity: a Two Step Process Mediated by 1
Dendrimer Exosite Binding
Introduction

1

Experimental Section

3

Results and Discussion

4

Conclusions

12

Bibliography

13

II. Nanoparticle Agglomeration Triggered by Protein Aggregation

16

Introduction

16

Experimental Section

18

vi

Results and Discussion

20

Conclusions

28

Bibliography

29

III. Multi-scale Anterior Cruciate Ligament Microdamage due to Normal 34
Mechanical Loading
Introduction

34

Experimental Section

35

Results and Discussion

37

Conclusion

40

Bibliography

40

Conclusions and Future Directions

43

vii

List of Figures
1.1

Synergistic exosite binding mechanism

2

1.2

G5 PAMAM and PEG FA-conjugates employed in this study.

5

1.3

Modified Stern-Volmer fitting of tryptophan quenching curves after the 6
conjugation

1.4

Locations of tryptophans (green and red) and bound FA (yellow) in hFR-

. 7

W171 & W102 (bottom) are highlighted in red.
1.5

Tight binding of G5Ac-COG-FA1.0 to FBP

9

1.6

Tryptophan fluorescence from FBP

10

1.7

Binding kinetics between G5Ac-COG-FA1.0 and FBP.

12

2.1

A target-ligand triggered biological cascade.

20

2.2

Agglomeration of ~40 nm FA-IOP triggered by FBP binding and aggregation 21
as measured by DLS

2.3

AFM image of the ~40 nm FA-IOP mixed with FBP.

2.4

Agglomeration of the ~450 nm FA-IOP triggered by FBP binding and 24
aggregation as measured by DLS.

viii

23

3.1

AFM height images of voids in ligament associated with mechanically 37
induced microdamage.

3.2

AFM-IR spectra from the ligament

38

3.3

Fibril void on ligament detected by SHG.

38

3.4

Collagen disordering detected by CHP-TAMRA.

39ix

Abstract
Drug conjugation gives many advantages such as improved targeting, solubility andretention. How conjugated drugs interact with proteins is a key question to answer intargeted drug delivery. This not only includes the interaction with the target protein, butalso includes the proteins that the drug carrier may encounter during drug trafficking. Folicacid (FA) is of wide interest as a targeting agent. It provides targeting effect towards thefolate receptor (FR), a commonly overexpressed protein in breast cancer and ovariancancer. Chapter one focused on developing a conjugated tighter binder towards the soluble
form of folate receptor, folate binding protein (FBP). This strategy employed a PAMAM
dendrimer conjugated folic acid; the dissociation constant was improved from 20 nM offree folic acid to 2 nM of the dendrimer conjugated folic acid. The folate binding protein
is a minor serum protein and is known to aggregate upon binding to folic acid. Thisaggregation could pose risk factors for drug delivery vectors such as the folic acidconjugated iron oxide nanoparticles. In chapter two, folate binding protein was shown toaggregate to large iron oxide nanoparticles that are conjugated to folic acid, triggering theagglomeration cascade that could drastically change the size of these nanoparticles.
Chapter three addresses a question that is important to sport medicine: what leads
to non-contact anterior cruciate ligament (ACL) failure? Our hypothesis was that beforexthe ACL fracture, microdamage would have already been created by jumping and landing
activities. In this chapter, paired cadaveric knees were collected and one of the pair wasmechanically tested under submaximal loading to see to what extent the microdamage
would be created. A hierarchical damage ranging from nanometer level to micrometer level
were detected using various methods including atomic force microscopy (AFM), nanoIR,second harmonic generation (SHG) and confocal microscopy. These microdamages
exhibits patterns that resemble the fractured ACL seen in the clinical patients.

xi

Chapter I
Enhanced Folate Binding Protein Avidity: a Two Step Process Mediated by
Dendrimer Exosite Binding
Introduction

Polymer-ligand conjugates are designed to bind protein active sites for applications
as drugs, imaging agents, and transport scaffolds.1-3 The polymer provides many potential
advantages including increasing ligand solubility, enhancing the circulation time, andshielding the ligand from unwanted protein interactions. These strategies have beensuccessfully implemented for mono-functional poly(ethyleneglycol) (PEG) polymers andtranslated into successful drugs in the clinic.4 In principle, polymer scaffolds can also be
employed to improve ligand to protein binding. However, the major strategy employed inthis regard, multivalent display of the ligand, has failed to realize the theoreticallyachievable improvements in binding.5-6
Vitamins are particularly attractive molecules for drug targeting since they arenaturally occurring, often exhibit low toxicity and high stability in plasma and tissue, andare preferentially taken up by quickly replicating cancer cells.7 Substantial effort has been
expended to develop polymer conjugates of vitamins for use in targeting and imaging
cancer cells. In particular, vitamin B9 or folic acid (FA) has received extensive attention
in this regard, and seven molecular conjugates have advanced to clinical trials. 7 Despite
great promise, FA-conjugates have not yet advanced to clinic. Polymer conjugates of FA
1

have also been extensively explored
including multivalent display of the

Figure 1.1 Synergistic exosite binding mechanism.
The –helix and –sheet secondary structure
formed upon FA binding is highlighted purple in the
X-ray crystal structure below.

ligand to enhance total binding strength
and/or selectivity to displays of FA
receptors.8-9
We now report a new strategy toachieve tight polymer-ligand conjugate
binding to target proteins by exploiting polymer-protein interactions. The strategy employs
substrate-triggered exosite binding of the protein and polymer surfaces. It is known that
FA induces changes in the secondary structure of FBP upon binding, including theformation of new domains of –helix and –sheet and rearrangement of a loop near theentrance to the binding site.10 We show that after these structural changes have been
triggered by binding, the generation five poly(amidoamine) (G5 PAMAM) dendrimer
conjugated to the FA interacts with the protein surface. This new exosite interaction resultsin strong binding of the ligand-dendrimer conjugate to FBP. The polymerconjugate/exosite binding strategy is potentially useful as it offers specificity of interaction
based on the natural protein ligand. This strong, tight binding could be of substantial benefit
for FA-conjugated imaging agents. Figure 1.1 illustrates the proposed binding mechanismwith the induced –helix and –sheet highlighted.
Folate binding protein (FBP) describes two membrane-bound isoforms of theprotein: folate receptor alpha (FR-) and folate receptor beta (FR-β) that are linked toplasma cell membranes via glycosylphosphatidylinositol (GPI) anchors.11-12 There is also
a secreted form, FR-γ, that lacks the GPI anchor. All forms of the protein have molecular2weights at around 30 kDa. Soluble FBP, present at 1-2 nM concentration in human serum
and other body fluids and 100 nM concentrations in milk,12-17 is believed to be a mixtureof FR-γ and FR- that has undergone cleavage of the GPI anchor. X-ray crystal structuresof FA-bound FR- and FA-bound FR-β were recently reported.10, 18 In the studies reported
here, we employed bovine FBP (bFBP) isolated from milk. bFBP has overall >80%
homology with human FR- (hFR-). In particular, there is 100% homology for 21 key
residues forming the FA binding pocket, which includes the –QSWRKERVL- section of
the inner strand of the nascent –sheet that forms upon FA binding.
Experimental Section
Materials. PEG-FA conjugates (2k, 5k, 30k) were purchased from Nanocs Inc. G5
PAMAM dendrimer was purchased from Dendritech and purified to remove trailinggeneration and G5 oligomer impurities as previously reported.19 G5Ac-COG-FA1.0 (credit:
Mallory A. van Dongen, Ananda Kumar Kanduluru, Philip S. Low)20 and G5Ac-FITC1.9FA4.4 (credit: Rachel L. Merzel, Casey A. Dougherty) were synthesized according topreviously reported methods.21-22 All dendrimers used have the primary amine terminalgroups acetylated (Ac) to avoid protonation in aqueous buffer. Whey protein concentrate
was purchased from Natural Foods Inc. and purified as briefly described below following
previously reported methods.23 All other chemicals and materials were purchased from
Sigma-Aldrich or Fischer Scientific and used as received.
Extraction, Purification, and Activity of FBP
A 200 mL bed volume of Sepharose 4B was activated by cyanogen bromide andsubsequently conjugated to FA. A pH 7.0 2% (w/v) whey protein solution was centrifuged3

(20,000 g) for 20 minutes. The resulting supernatant was loaded on the FA affinity column.
10 L of supernatant was run through the column prior to the onset of back-pressure.
Unbound protein was washed away using 2 L of 1 M NaCl followed until the solution ran
clear using at least 2L of nanopure water. 300 mL of 0.3 M acetic acid solution was used
to flush the FBP off the column. The solution was collected by fractions. Fractions with
90% FBP purity were combined and the pH was adjusted to 7.0 by the addition of 5.0 M
NaOH.
Fluorescence Measurements
All fluorescence measurements were carried out on a Varian Cary Eclipse
Fluorescence Spectrophotometer. A 58 nM folate binding protein solution was made in pH
7.4 1x PBS. The temperature control was set at 22 °C. Excitation wavelengths of 280 nm
for tryptophan and 495nm for FITC were employed. The slit widths were 8 nm for both
excitation and emission monochromators. In all cases, fluorescence measurements weretaken after the solutions had reached an equilibrium value.
Results and Discussion
In previous surface plasmon resonance (SPR) studies of G5-FA/FBP binding, we
discovered that G5-FAn (n = 1, 2 or various navg) bound essentially irreversibly to bFBP.20,
24

Using atomic force microscopy (AFM) force-pulling studies, we found that the mostprobable rupture forces were 83, 201, and 189 pN for G5-FA2.7, G5-FA4.7, and G5-FA7.2,
respectively.25 Intriguingly, although the probability of interaction decreased with FAblocking, the force magnitude of the most probable rupture force increased from 189 to
274 pN for G5-FA7.2. The combination of the SPR and AFM force-pulling data is

4

Figure 1.2. G5 PAMAM and PEG FA-conjugates employed in this study.

G5Ac-COG-FA

G5 PAMAM~5nM

Dipeptide Linker(Asp, Lys)FA

COG linker(cyclooct-1-yn-3-glycolic acid)

G5Ac-FITC1.9-FA4.4

PEG(30k)-FA, n=671
PEG(2k)-FA, n=34
G5 PAMAM

FA

consistent with a slow-onset, tight-binding (SOTB) FA/FBP binding mechanism whereFA-binding induces significant structural change in the bFBP secondary structure,
allowing a network of van der Waals interactions to form between the G5 PAMAM
dendrimer surface and the bFBP surface. 20, 25
The interaction between polymer backbones and proteins is considered to be themost important factor in polymer-based drug delivery and biodevice development.26
Proteins have been shown to interact with polymers via a complex set of interactions that
include generalised electrostatic,27 van der waals,28 and H-bonding29 interactions and more
specific protein-polymer binding.30 These interactions have generally been studied in thecontext of protein adsoprtion onto surfaces as opposed to the triggered exosite binding
described herein.
To further explore the nature of these interactions, we have performed a series ofsolution-phase fluorescence quenching experiments taking advantage of the intrinsic
fluorescence associated with the 11 tryptophan residues present in bFBP. For these studies,5we employed the following FA-polymer conjugates: 1) FA stochastically conjugated to aterminal PAMAM amine group (Figure 1.2, G5Ac -FITC1.9-FA4.4). This provides a directcomparison to our initial SPR24 and force-pulling25 studies and other stochastic FA
conjugations for G5 PAMAM reported in the literature;8, 31-34 2) A G5-FA conjugatecontaining precisely one FA molecule per G5 PAMAM polymer (G5Ac-COG-FA1.0). Thiseliminates any effects arising from multivalent FA interactions including bridging between
two bFBP. Recent SPR studies on G5Ac-COG-FA1.0 led to the proposed SOTB interaction
for G5-FA/FBP binding;20 3) FA-PEG conjugates directly linked via a FA carboxylic acidto the terminal PEG amine. The 30 kDA PEG (PEG30k-FA) was chosen to provide anequivalent MW to that of acetylated G5Ac PAMAM. The 2 kDa PEG (PEG2k-FA) waschosen to provide a PEG conjugate with less steric bulk.
Fluorescence Quenching of bFBP upon FA and FA-conjugate Binding
Fluorescence quenching titration
provides a high sensitivity method forstudying biological processes due to its
highsensitivity.35

Howeverwhen

Figure 1.3. Modified Stern-Volmer fitting oftryptophan quenching curves after the conjugation.
G5Ac,
PEG2k-COG-FA(mix) ,PEG30k-FA,
PEG2k-FA,
G5Ac-FITC1.9-FA4.4,
FA,
G5Ac-COG-FA.
The concentration of FBP in each case is 58 nM (pH
7.4, 1x PBS solution).

employing this technique to makequantitative

measurements,

itisimportant to correct for inner filter
effects (IFE) and dynamic quenchingthat can decrease the observed light6

Figure 1.4. Locations of tryptophans(green and red) and bound FA (yellow)
in hFR-. W171 & W102 (bottom) arehighlighted in red.

intensity.36 At the submicromolar concentrations oftitrant employed for these studies, IFE did notcause an observable effect in light intensity for anyof the polymer conjugates.

Titration of a 58 nM FBP solution with FA
results in a roughly 50% quenching of the native
tryptophan fluorescence (Figure 1.3). This experiment was suggested by Dr. E Neil G
Marsh. A comparison of the apo-FPB and FA-FBP X-ray structures indicates that the
majority of the tryptophan residues do not change position upon FA binding.10 Notable
exceptions are W171, which is located on the protein surface away from the FA binding
site, and W102, which is located on the loop adjacent to the FA-binding site and plays a
key role in FA binding and –sheet formation (Figure 1.4). The distance between W102
and the FA benzamide ring (5.1 Å, 15°angle) is consistent with parallel-displaced -stacking.10 Tryptophan W171 is also of particular interest as it -stacks with FA pteridine
ring (3.7 Å, 15°angle). W64, 120 134, 138, and 140, all located along the FA binding site,
are highlighted in green in Figure 1.4. Titration with G5Ac-FITC1.9-FA4.4 resulted in asimilar quenching level to that observed for free FA (Figure 1.3). Surprisingly, titration
with G5Ac-COG-FA1.0 resulted in roughly 80% fluorescence quenching and exhibits asteeper initial slope indicating a different stoichiometry of interaction. Treatment of FBP
with free FA, followed by addition of acetylated G5 dendrimer (G5Ac), resulted in no
additional fluorescence quenching. This result indicates that for the enhanced fluorescence7

quenching to occur the dendrimer must be conjugated to FA. Thus, synergistic binding
requires both FA-induced FBP structural change and FA conjugation to the dendrimer.
In order to explore the role of polymer in the binding process, studies were carriedout with PEG30k-FA and PEG2k-FA. As illustrated in Figure 1.3, titration of FBP with
PEG30k-FA or PEG2k-COG-FA resulted in only about 20% fluorescence quenching and had
not yet reached saturation by 120 nM of added conjugate. This data demonstrates theequilibrium binding shift expected for conjugates that have steric constraints to achieving
FA-FBP binding. The PEG2k-FA exhibits similar quenching to free FA whereas PEG30kFA requires a roughly 2-fold greater concentration to achieve saturated binding. PEG2kCOG-FA binds less effectively than PEG2k-FA suggesting that the COG linker isincreasing steric constraints in this case. This is an important control as it demonstrates
that the presence of the triazole ring/tryptophan interactions do not induce the large amountof quenching observed for G5Ac-COG-FA1.0. The PEG-FA conjugates demonstrate thetypical result of polymer conjugation to a protein binding substrate, namely, equivalentbinding or reduced binding caused by steric constraints. The results provide a distinct
contrast to the binding interaction of G5Ac-COG-FA1.0 with FBP where a dramatic increasein binding constant20 and fluorescence quenching is observed.
The fluorescence quenching curves were fit to the modified Stern-Volmer equation
to assess binding avidity of polymer conjugates to FBP. 37
f [Q]I 1 aI0
K D  [Q]8

Figure 1.5. Tight binding of G5Ac-COG-FA1.0 to FBP. A) Fluorescence quenching of 58 nM FBP in thepresence of 800nM FA B) 80 nM G5 Ac-COG-FA1.0 binds to FBP, displacing FA, in the presence ofexcess FA. C) The presence of an additional 800nM excess FA does not displace G5 Ac-COG-FA1.0.
The FBP concentration is 58 nM (pH 7.4, 1x PBS solution).

I is the intensity of the fluorophore, fa is the fraction of accessible fluorophores, KD is thedissociation constant, and [Q] is the free titrant concentration calculated from a quadratic
equation (Scheme S1).38 A static quenching effect from binding was observed from thefitting. The plots (Figure 1.3) yield KD = 27.9 ±3.3 nM for FBP binding FA, similar to theliterature value of 20 nM,39 whereas the G5Ac-COG-FA1.0 conjugate bound FBP with KD =2.43 ± 0.17 nM, roughly ten-fold more tightly than FA. The PEG30k-FA isotherm boundwith KD = 51.4 ± 6.4 nM, indicating that the polymer plays a relatively small role inmodulating the binding avidity to FBP. G5Ac-COG-FA1.0 quenches a larger fraction ofaccessible fluorophores (fa = 87 ±1%) as compared to FA (fa = 67 ±3%) or PEG30k-FA (fa= 66 ± 4%). This is presumably an indication of the large conformational change inducedby the dendrimer binding to the exosite on the protein surface.
The binding avidity comparison of FA/FBP and G5Ac-COG-FA1.0/FBP was tested
by first saturating FBP with 14 equiv FA (Figure 1.5). The subsequent addition of 1.4
9Figure 1.6 Tryptophan fluorescence from FBP a)
Tryptophan fluorescence from FBP in the presence offolic acid (FA) and G5Ac-FITC1.9-FA4.4 conjugate, ex:
280nm b) FITC fluorescence from G5 Ac -FITC1.9-FA4.4
in the presence of folic acid (FA) and FBP, ex: 495nm.
The concentration of FBP in each case was 58 nM (pH
7.4, 1x PBS solution).

a)Intensity

additional quenching to the 80% level
previously observed for G5Ac-COGFA1.0 alone. We interpreted this as

FA by G5Ac-COG-FA1.0 in the FBPFree FBP

binding

300

pocket100

the10-fold

The addition ofanother 14 equivalents of FA, to give

0
300

320

340

360

380

a

400

Wavelength (nm)28-fold

dendrimer

excessofconjugate,

FA

over

failedto

100nM G5Ac-FITC1.9-FA4.4

displace G5Ac-COG-FA1.0 from FBP.

100nM G5Ac-FITC1.9-FA4.4+58nM FBP
400

bystronger binder.

200

b)500

induced

100nM G5Ac-FITC1.9-FA4.4+58nM FBP
100nM G5Ac-FITC1.9-FA4.4+58nM FBP + 100nM FA

400

G5Ac-COG-FA1.0

essentially complete displacement of

100nM G5Ac-FITC1.9-FA4.4
500

equiv

100nM G5Ac-FITC1.9-FA4.4+58nM FBP + 100nM FA

IntensityThus, the COG linked conjugation of
300

200

FA to G5 PAMAM successfully

100

created a stronger binder to the FBP

0
520

540

560

580

protein that acts via dendrimer

600

Wavelength (nm)binding to the FBP surface outside of
the FA binding pocket, i.e. an exosite binding interaction. As illustrated in Figure 1.1, thenearest region showing extensive FBP surface structural change is the –sheet region andthis appears to be likeliest dendrimer/protein binding site. However, the 30 kDa dendrimer
is similar in size to FBP and binding to the induced -helix or other protein surface sites
cannot be ruled out. For this system, a synergistic interaction between the dendrimer andthe protein affords tight binding.

10We also explored competitive binding of FA/FBP and G5Ac-FITC1.9-FA4.4/FBP. In
this instance, the tryptophan fluorescence and FITC fluorescence can be observed inseparate channels. Both tryptophan and FITC fluorescence are quenched upon mixing 100
nM G5Ac-FITC1.9-FA4.4 with 58nM FBP (Figure 1.6, red curves). Upon addition of 100 nM
FA, a small amount of additional quenching is observed consistent with the initial substoichiometric treatment of FBP with G5Ac -FITC1.9-FA4.4 (Figure 1.6a, blue curve). Thisevidence, coupled with strong quenching observed by G5Ac-COG-FA1.0, provides
additional support for the conclusion that the multiple FAs present on G5Ac -FITC1.9-FA4.4
do not generate additional FBP quenching, and therefore binding, and that the multiple FA
ligands are not generating interactions with multiple proteins. On the other hand, the FITC
fluorescence increases upon addition of excess FA. This suggests that G5Ac-FITC1.9-FA4.4
is bound more weakly than G5Ac-COG-FA1.0 as it is displaced by excess FA. The SternVolmers analysis of G5Ac -FITC1.9-FA4.4 showed this material quenches a similar fractionof accessible fluorophores (fa= 57 ± 2%) as compared to FA (fa = 67 ±3%) with a slightly
smaller dissociation constant of KD = 16.0 ±1.9nM as opposed to KD = 27.9 ±3.3nM fromFA.
The G5Ac-COG-FA1.0/FBP binding kinetics were studied under pseudo-first orderconditions by mixing 1-20 nM G5Ac-COG-FA1.0 into a 58 nM FBP solution. The
fluorescence changes were followed over 1-2 hours until equilibrium was reached (Figure
1.7 inset). The resulting curves were fit to single exponentials to obtain kobs for eachconcentration. kobs was fit as a function of G5Ac-COG-FA1.0 concentration to the equation
below that describes a two-step tight binding SOTB model. In the equation, we use P* to11

indicate a change in protein structure, a common feature of the SOTB model, and L* toindicate the change in dendrimer structure as highlighted in Figure 1.1.

P + L

k-1

kobs 

k2

k1

P-L

k-2

P*-L*

k2 [ L]+k2
K D  [ L]The fit provided a KD value of 9.9  8.5 nM, a k2 value of 0.07  0.02 s-1, and a k-2

value of 0.008  0.007 s-1 (Figure 1.7). Recall the KD value obtained from the modified
Stern-Volmer analysis was 2.4 ±0.2nM. These two values agree well with each other. The
rate constant k2 indicates a slow onset binding interaction between the dendrimer andprotein.
Conclusions
Substrate-triggered polymer
binding to an FBP exosite gives anorder of magnitude greater binding

Figure 1.7. Binding kinetics between G5Ac-COG-FA1.0 and
FBP. kobs values were obtained by fitting the curves
obtained for the addition of 1, 2, 5, 10, and 20 nm
solution of G5Ac-COG-FA1.0 to 58 nM FBP (pH 7.4, 1x PBS
solution). Inset: Change in fluorescence over time for
addition of 2nM G5Ac-COG-FA1.0.

constant than that obtained by thenative FA ligand. In addition, FAtriggered exosite binding is not
displaced by presence of a 28-foldexcess of free FA. Tight polymerprotein binding, triggered by specific12ligand binding, provides promising strategy for binding scaffolds containing drugs and/or
imaging agents to desired protein targets.
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E.; Melcher, K., Structural basis for molecular recognition of folic acid by folate receptors.
Nature 2013, 500, 486-490.
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Kamen, B. A., Folate Receptors and Therapeutic Applications. In Targeted Drug
Strategies for Cancer and Inflammation, Jackman, A. L.; Leamon, C. P., Eds. Springer:
2011.
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Holm, J.; Babol, L. N.; Markova, N.; Lawaetz, A. J.; Hansen, S. I., Theinterrelationship between ligand binding and thermal unfolding of the folate binding
protein. The role of self-association and pH. BBA-Proteins Proteom 2014, 1844 (3), 512519.
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Holm, J.; Bruun, S. W.; Hansen, S. I., The Complex Interplay Between Ligand
Binding and Conformational Structure of the Folate Binding Protein (Folate Receptor):
Biological Perspectives. BBA-Proteins Proteom 2015, 1854, 1249-1259.
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Wibowo, A. S.; Singh, M.; Reeder, K. M.; Carter, J. J.; Kovach, A. R.; Meng, W.
Y.; Ratnam, M.; Zhang, F. M.; Dann, C. E., Structures of human folate receptors reveal
biological trafficking states and diversity in folate and antifolate recognition. Proc. Natl.
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van Dongen, M. A.; Desai, A.; Orr, B. G.; Baker, J. R.; Banaszak Holl, M. M.,
Quantitative Analysis of Generation and Branch Defects in G5 Poly(amidoamine)
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van Dongen, M. A.; Silpe, J. E.; Dougherty, C. A.; Kanduluru, A. K.; Choi, S. K.;
Orr, B. G.; Low, P. S.; Banaszak Holl, M. M., Avidity Mechanism of Dendrimer-Folic
Acid Conjugates. Molec. Pharm. 2014, 11, 1696-1706.
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Holl, M. M. B., Design, Synthesis, and Biological Functionality of a Dendrimer-Based
Modular Drug Delivery Platform. Bioconjugate Chem. 2011, 22 (4), 679-689.
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Measurement of Binding Constants by Frontal Analysis Continuous Capillary
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Browne, M. M.; Lubarsky, G. V.; Davidson, M. R.; Bradley, R. H., Protein
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Silpe, J. E.; Sumit, M.; Thomas, T. P.; Huang, B.; Kotlyar, A.; van Dongen, M.;
Banaszak Holl, M. M.; Orr, B. G.; Choi, S. K., Avidity Modulation of Folated-Targeted
Multivalent Dendrimers for Evaluating Biophysical Models of Cancer Targeting
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Sunoqrot, S.; Bugno, J.; Lantvit, D.; Burdette, J. E.; Hong, S., Prolonged blood
circulation and enhanced tumor accumulation of folate-targeted dendrimer-polymer hybrid
nanoparticles. J. Controlled Rel. 2014, 191, 115-122.
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tumor cells for lysis. Proc. Natl. Acad. Sci. 1995, 92 (20), 9057-9061.

15

Chapter II
Nanoparticle Agglomeration Triggered by Protein Aggregation
Introduction
Folate receptor-targeted nanoparticles have been widely employed as a tumor
targeting strategy for imaging and drug delivery applications.1-10 Conjugation of folic acid(FA) to small molecule drugs and nanoparticles provides a convenient, cheap, andchemically stable cancer targeting vector. Despite substantial promise and seven clinical
trials to date,11 neither molecular nor nanoparticulate FA conjugates have reached the clinic,
suggesting challenges to their use in vivo. Herein, we focus on interactions of FA withserum folate binding protein (FBP) and how these interactions may impact the use of FA
as a targeted delivery agent.
It is well established that FA induces the aggregation of several proteins in serum,
notably albumin, lactoferrin, and FBP.12-18 We have previously noted that FA-dendrimer
conjugates can lead to strong binding interactions between the polymer and FBP surface19
and that FA, methotrexate (MTX), and leucovorin all increase the aggregation propensity
of FBP.18 Furthermore, FA- and MTX-polymer conjugates induce substantially more
fluorescence quenching of FBP than FA or MTX alone, indicating that the conjugates areaffecting protein structure and potentially aggregation behavior.20 For these reasons, we
decided to explore the role of FA-induced FBP aggregation in causing the agglomerationof FA-conjugated nanoparticles. The formation of agglomerated particles and the masking16

of the conjugated FA by FBP binding is likely to play an important role in transport andother in vivo behavior of FA-conjugated materials.
For this initial study we selected the well-studied class of iron oxide particles (IOP)
that have been explored for imaging and drug delivery applications.21-24 IOPs have been
extensively investigated for imaging and drug delivery due to their low toxicity,
biocompatibility, large surface area, and suitable magnetic properties. IOP-based agents inthe 30 – 300 nm diameter size range have been approved for clinical use, and additional
materials are currently in clinical trials.25 Particles under consideration have sizes ranging
from 20 nm to 3500 nm in diameter.26 The majority of them are administered via
intravenous injection. However, it is known that IOPs, especially unmodified ones, areprone to agglomeration and even precipitation in physiological environment.27 Therefore,
investigation of IOP agglomeration in the presence of serum proteins is crucial whenevaluating the safety and efficacy of these nanoparticles.
Folic acid conjugated iron oxide nanoparticles were synthesized to study particle
agglomeration in the presence of FBP and albumin (Figure 2.1). It was found that selfaggregation of FBP upon FA binding further triggers the agglomeration of the IOP particles.
Considering the drastically smaller size of protein compared to the iron oxide particles, thisaggregation-agglomeration event resembles a biological cascade – a small initial event(ligand-protein binding) leading to larger effects (protein structural rearrangement), thatthen leads to protein aggregation and particle agglomeration. The agglomerates formed areorders of magnitude larger in size than the initiating small molecule. Two iron oxide-FA
conjugates, a ~40 nm FA-IOP and a ~450 nm FA-SeraMag, were selected for use in thisstudy to model the range of particle sizes applied in the clinic. In particular, the ~40 nm
17

IOP represents a class of SPIONs (superparamagnetic iron oxide nanoparticles) that is acommonly used nanoparticle platform for drug conjugation and the development oftargeted theranostics.28-30 The ~450 nm SeraMag is a commercially available particle
widely used in protein separation and preparation.31-33 The two IOPs allow us to contrast
the behavior of particles designed for very different applications, which highlights bothdifferent surface chemistry and size range.
The FA-conjugated beads were mixed with human serum albumin (HSA) and folate
binding protein (FBP) at the physiological concentration of these serum proteins. At 2 nM
FBP with 0.01% w/w 40 nm particles in 1x PBS buffer (28 nM nominally), the cascade
resulted in agglomeration to form 3800 ± 1100 nm diameter particles. Similarly, at 2 nM
FBP with 0.01% w/w 450 nm particles (SeraMag) in 1x PBS buffer (16 pM nominally),
agglomeration resulted in 4030 ±1100 nm diameter particles. Albumin, the most abundant
serum protein, had no significant impact on ~40 nm FA-IOP particle agglomeration up to
the micromolar level; however, it did inhibit the agglomeration of the ~450 nm FASeraMag particles. In addition to dynamic light scattering (DLS), atomic force microscopy(AFM) was carried out to study the morphology of agglomerated particles.
Experimental Section
Materials The ~450 nm amine-blocked iron oxide beads were obtained from GE
healthcare (Sera-Mag SpeedBeads Amine-Blocked Particles, 10 mg/mL). The ~40 nm ironoxide particles functionalized by polyethylene glycol(PEG-2k) with amine terminal groups
were obtained from Ocean NanoTech. All other chemicals were purchased from Sigma
Aldrich if not otherwise specified. The folate binding protein was isolated and purified aspreviously reported.2
18

Folic acid conjugation to magnetic beads
Folic acid was conjugated to the beads via an EDC-NHS carbodiimide coupling
procedure. Specifically, 300 L MilliQ water, 34 L folic acid solution (3.9 mg dissolvedin 1 mL DMSO, then diluted 100x by MilliQ water), 51.7 L NHS solution (17.9 mg NHydroxysuccinimide in 100 mL MilliQ water), 35.9 L EDC solution (4.0 mg 1-Ethyl-3(3-dimethylaminopropyl) carbodiimide in 100 mL MiliQ water) and 578.4 L MilliQ water
were added into a vial for carboxylate activation. The vial was transferred to a shaker (23C, 115 rpm) for 7h (credit: Sarah Klem, Alexis K Jones).
1% Magnetic bead stock solutions containing 0.05% sodium azide were vortexed
and 1 mL was pipetted into a vial and placed on a magnet for about 3 min. The stock buffer
was discarded and the beads washed with MilliQ water twice. The beads were re-suspended
into the activation solution described above and allowed to react for 12 hrs. The beads
were then washed with water and characterized by IR spectroscopy.
AFM experimentProtein solutions are made in filtered 1x PBS at their corresponding physiologicalconcentration: 2 nM for FBP. The 1% conjugated beads stock solution was added into theprotein solution to make a final concentration of 0.01% w/w. 40 L of the resulting solution
was spin coated at 4,200 rpm onto a freshly cleaved mica surface. The sample was further
rinsed with another 40 L milliQ water while spinning to rinse any remaining salt from thesurface. PBS salts crystals can occasionally be found on the mica surface but they havedramatically different morphology from the IOP particles. AFM imaging was carried out
in tapping mode using PicoPlus 5500 (Agilent Electronic Measurement, Aspire CT300R
19tip) and NanoIR-2 (Anasys, Anasys PR-EX-T125-10 tip) AFM systems. For the ~ 40 nm
FA-IOP the particle size is similar to the nominal 10 nm tip radius. Tip deconvolution was
done to better estimate the particle diameter (d=dc2/8R, where d is the particle diameter, dc
is the observed diameter, and R is the tip radius).
DLS experiment
The sample solutions were prepared as describe for the AFM experiments. DLS
was performed on a Nanosizer ZS (Malvern Instruments Ltd.) instrument. The DLS in
figure 2.2 and figure 2.4 was taken right after FBP addition into the solution. Additional
DLS experiments on agglomerate particle stability were also taken under same conditions(Figures S7). The solutions were measured in a 1 mL cuvette at 25 °C. The refractive index
and absorption coefficient for the particles were set at 2.42 and 0.01, respectively. All data
is an average of 15 scans.
Results and Discussion
Folic acid modification on IOP surfaces has widely been used as a targeting strategy
in drug development. The surface modification of folic acid on IOP may influence theinteractions of the particles with serum proteins in a nonspecific fashion due to its

Figure 2.1. A target-ligand triggered biological cascade. Addition of a physiological concentration of
folate binding protein (2 nM FBP) triggers the agglomeration of the large folic acid conjugated ironoxide particles in 1x PBS, even with the presence of a high concentration of human serum albumin.

20

Figure 2.2. Agglomeration of ~40 nm FA-IOP triggered by FBP binding and aggregation as measured by
DLS: a) 0.01% w/w FA-IOP + 2 nM FBP followed by addition of HSA. b) 0.01% w/w FA-IOP + HSA
followed by addition of 2 nM FBP.

hydrophobicity and in a specific fashion due to FA-FBP interactions. Both of theseinteractions have the potential to lead to particle agglomeration in the blood serum.
FBP and albumin are the proteins primarily responsible for the binding andtransportation of folates in blood serum.34 FBP exists at a 1-2 nM concentration in humanserum whereas the much more abundant human serum albumin (HSA) is present at 30
μM.35 FBP binds to FA tightly with a nanomolar dissociation constant, and the protein selfaggregates upon substrate binding.2, 14 Crystallographic studies indicate that FBP forms abeta sheet upon folic acid binding and it is hypothesized that this contributes to theaggregation of the protein. 20 This observation led us to wonder if the protein-protein
aggregation could also trigger the agglomeration of large FA conjugated particles. FBP
aggregation is stable at pH 6-9 and at temperatures ranging from 20°C to 37°C.16, 36 HSA
is a ~40,000 times weaker binder for folate as compared to FBP.37 However, it composes
more than half of total serum proteins and is also involved in folate transportation andregulation in blood.

21

In this study, two iron oxide particles are coated with FA via EDC/NHS coupling
as a model system for folic acid-targeted nanomedicine. The particles were characterizedusing infrared spectroscopy. The ~40 nm FA-IOP IR spectrum has peaks at 2916 cm-1 and2851 cm-1, which correspond to the CH2 stretching vibrations for the polyethyleneglycol(PEG) surface coating on iron oxide nanoparticles.38 An FA carbonyl stretch, 1646 cm-1
for free FA, is observed at 1632 cm-1 for conjugated FA-IOP. A strong peak at 983 cm-1 isalso indicative of FA and shifts to 1004 cm-1 upon conjugation. The FA-SeraMag showeda peak at 1560 cm-1 in the IR spectrum, which is also detected in the unconjugated SeraMag
IR spectrum. An FA carbonyl stretch appears at 1654 cm-1 in the conjugated particle andappears to provide an increase in intensity in that region. A strong peak at 989 cm-1 is also
observed for conjugated material.
DLS and AFM were employed to study the agglomeration of particles induced byserum protein binding. A 0.01% w/w FA- conjugated nanoparticle (FA-IOP and FASeraMag) solution was mixed with serum proteins at their physiological concentration. All
the solutions were made in 1x PBS buffer to mimic the salt environment in blood serum.
The 0.01% w/w (28 nM nominally) ~40 nm FA-IOP aqueous 1X PBS solution yielded one
peak with a number weighted average diameter at 39 ±13 nm (Figure 2.2a). Upon mixing
with 2 nM FBP, a peak centered at 3800 ± 1100 nm was detected. The resulting
agglomerate is nearly 100-fold larger in diameter (106 larger in volume) than the original
particle. Addition of up to 6 µM HSA into this solution did not have a significant impact
on the DLS signal. This process was also studied in reverse: HSA was added to the FAIOP solution prior to FBP addition. At 30 µM HSA, the particles have a measured diameterof 42 ± 13 nm (Figure 2.2b). The hydrodynamic size of HSA monomer is also detected22under this condition at 7.0 ± 1.4 nm, which is in agreement with the literature value.39, 40
These data indicate the HSA non-specific binding on this particle is minimal. Addition of
2 nM FBP into this FA-IOP/HSA solution generated agglomerated particles with adiameter of 3100 ± 850 nm, and zeta potential of 4.7 ± 0.3mV. These data indicate that
HSA does not trigger a cascade and that excess HSA does not inhibit particle
agglomeration upon the addition of FBP.
In order to obtain information about particle morphology, the solutions were spincoated onto a cleaved mica surface and characterized using tapping mode AFM (Figure2.3). The 0.01% w/w FA-IOP particles alone demonstrated a well-dispersed nonagglomerated pattern on the mica surface with an average diameter of 59 ± 7 nm (Figure

Figure 2.3. AFM image of the ~40 nm FA-IOP mixed with FBP. a) 0.01% w/w FA-IOP without FBP b) A
typical agglomerated particle at 0.01% w/w FA-IOP + 2 nM FBP c) Line scan profile of theagglomerated particles. The particle surface can be sectioned into ~50 nm spheres d) 3D rendering ofan agglomerated particle.

23

2.3a). This diameter was corrected to 44 ± 10 nm after tip deconvolution. FBP-assisted
particle agglomeration was observed by AFM once the 0.01% w/w FA-IOP particle was
mixed with 2 nM FBP. An average of five agglomerates yielded a particle volume of 58 ±1.9 μm3 (equivalent to ~210,000 ~40 nm FA-IOP particles), corresponding to an average
diameter of ~ 2400 ± 800 nm. The particle diameter falls into the size range measured by
DLS (Figure 2.3b). The large particle agglomerates featured a rough surface consisting ofdistinct particles and line scan profiles demonstrated the existence of ~50 nm particles on
the agglomerate surface (Figure 2.3c). The size of the smaller components in the line scanis consistent with the size of individual FA-IOP particles. A 3D rendering of the aggregatedparticle is illustrated in Figure 2.3d. The small FA-IOP particles on the surface can beeasily seen in the large agglomerated particle. For the HSA containing samples, the high
HSA concentration resulted in a multi-layer protein coating on the mica surface and thisprevented measurement of the IOP aggregates.

24

The much larger FA-SeraMag particle (450 ±70nm) was also titrated with FBP and
HSA under similar conditions (Figure 2.4a). The addition of 2 nM FBP to form a 0.01%
w/w solution (16 pM nominally) shifted the entire peak to 4030 ±1100 nm with a measured
zeta potential of 1.5 ± 0.2 mV. The original peak for the 450 nm particles was no longer
observable. This indicates a full agglomeration of particles in the presence of 2 nM FBP.
This full shift of the peak is most likely due to the much higher protein to particle molarity
ratio in this case. For each ~450 nm FA-SeraMag particle there were 125 FBP protein
added whereas for each ~40 nm FA-IOP there were just 0.07 FBP (i.e., 14 IOP per FBP).
On the other hand, 600 µM (40 g/L) HSA lead to a two-fold increase in diameter (880 ±360 nm). This could be due to nonspecific binding from HSA and formation of a protein

Figure 2.4. a) Agglomeration of the ~450 nm FA-IOP triggered by FBP binding and aggregation as
measured by DLS. b) AFM image of 0.01% w/w FA-SeraMag mixed with 2nM FBP. c) Line scan profileof the agglomerated particles. The particle surface is composed of ~500nm spheres d) 3D image of theagglomerated particles.

25

corona at this concentration. Addition of 2 nM FBP into this solution did not introduce afull shift of this ~880 nm particle to a large agglomeration as was the case without HSA.
However, the peak associated with the ~880 nm particle exhibits tailing (Figure 2.4a, pink)
up to a diameter of ~3200 nm. It is worth noting that in the presence of HSA only a small
fraction of FA-SeraMag particles shifted to the large agglomeration and this differs
substantially from the addition of FBP alone. This leads us to hypothesize that the HSA
protein corona could be protecting the FA-SeraMag particles from agglomeration. The FBP
assisted particle agglomeration was measured by AFM for solutions containing 0.01% w/w
FA-SeraMag particle mixed with 2 nM FBP (Figures 4b, S5). The large agglomeratefeatured a rough surface and the line scan profile demonstrated the existence of ~500 nm
particles (Figure 2.4c). This result indicates that the large particle agglomerate is composedof the smaller FA-SeraMag components. A 3D rendering of the agglomerated particle isalso shown in Figure 2.4d. In this case, the average agglomerate contains ~360 FASeraMag particles. A summary of DLS size measurement for both IOPs is provided inTable I.

26

Table I. Number weighted average particle diameter from DLS
Sample# weighted average # weighted average
Ave. Dia. Peak 1 Ave. Dia. Peak 2/nm/nm
0.01% w/w FA-IOP
39 ±13
0.01% w/w FA-IOP
39 ±13+ 2nM FBP
0.01% w/w FA-IOP
7.3 ±1.5
42 ±13+ 30 µM HSA
0.01% w/w FA-IOP
7.0 ±1.4
54 ±10+ 30 µM HSA+ 2nM
FBP
0.01%
w/w
FA- 450 ±70
SeraMag
0.01%
w/w
FA- SeraMag+ 2nM FBP
0.01%
w/w
FA- 6.4 ±1.0
880 ±360
SeraMag+ 600µM HSA
0.01%
w/w
FA- 6.3 ±0.9
840 ±330
SeraMag+ 600µM HSA+ 2nM
FBP#
weighted
average Dia. Peak
3 /nm
3800 ±1100
3100 ±850

4030 ±1100--

The FBP-induced agglomeration of the ~40 nm and ~450 nm IOP resembles abiological cascade in two ways. First, there are three discrete steps (ligand binding, protein
structural change, protein self-aggregation) preceding the fourth step (particle
agglomeration). Second, the initial interaction occurs on a small scale (small moleculeinteraction with the protein binding pocket). This small interaction triggers a series ofchanges that results in agglomeration of the much larger ~40 nm and ~450 nm IOP.
Under the physiological concentrations employed, HSA is in large excess to the
IOPs. However, HSA does not bind to FA as tightly as FBP. The combination of these two
factors results in a substantial amount of free HSA left in solution. The non-specific binding27

of HSA to the surface conjugated FA does not introduce a structural change in HSA that
can induce IOP agglomeration. However, addition of 2 nM FBP into the ~40 nm IOPHSA solution does generate particle agglomeration. This shows that physiologicalconcentrations of FBP trigger can particle agglomeration even in the presence of the large
concentration of HSA found physiologically. HSA on the particle may make the surface
less accessible to FBP and provide some protection to IOP particle from agglomeration andthis is observed for the ~450 nm IOP where there is a much higher HSA concentration perparticle.41
Particle size can have a significant impact on tissue distribution in vivo. Particles
larger than 2000 nm accumulate readily in the spleen and liver. Particles ranging from 100
nm and 200 nm have been shown to escape liver and spleen filtration, while small
nanoparticles are filtered out by the kidneys.42 For the iron oxide particles in this study,
physiological concentrations of FBP triggered the agglomeration of FA-IOP in thepresence of HSA and altered them from ~ 40 nm diameter particles that could readilytransport through tissue into ~3000-4000 nm diameter particles that would be incapable oftissue penetration and would be trafficked to spleen and liver. The result illustrates theimportance of considering serum protein-induced particle agglomeration when designing
FA-targeted systems.
Conclusion
Nano-engineered drug delivery platforms have received great attention in the past
decade as therapeutic and diagnostic tools.3, 43-49 In order to best understand the safety andefficacy of these targeted nanomaterials in vivo, previous studies have explored theinteractions with serum proteins.41, 50-52 It is widely proposed that the absorbed protein layer28

on nanomaterials influences the bio-identity and therein the transportation and uptake ofthe nanomaterial into target cells.53-56 Future in vitro studies of the nanomaterial interaction
with serum proteins, and the resulting effect on cellular uptake, will play an essential rolein understanding the pathology of these materials. In the work described here, folic acidconjugated iron oxide nanoparticles, initiate a set of interactions with serum proteins that
leads to particle agglomeration. Additional work is needed to fully understand theimplications of this agglomeration for biological trafficking and potential health risks.57-63
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33

Chapter III.
Multi-scale Anterior Cruciate Ligament Microdamage due to Normal Mechanical
Loading
Introduction
The anterior cruciate ligament (ACL) is a key connective tissue stabilizing the knee.
Over 200,000 ACL injuries occur annually in the US1. The lifetime public health burdenof ACL tears in the US was estimated to be $7.6 billion annually when treated withreconstruction and $17.7 billion annually when treated with rehabilitation.1 The number of
ACL reconstructions in the US is growing rapidly with an estimated annual increase
between 3.4% and 7.7%.2-4 Among them, nearly three quarters of ACL injuries follow a
non-contact mechanism even in the high contact sport of NFL football.5 However, thecause of non-contact ACL injuries remains somewhat of an enigma. Several theories havebeen proposed to explain the mechanism of non-contact ACL injury, including aggressive
quadriceps loading,6 excessive joint compressive loading,7 awkward landing or
decelerating gestures,8 and neuromuscular control deficit.9
Current orthopedic studies focus mainly on the acute triggers of the injury, per the
traditional view of the non-contact ACL injury. Very few studies have been conducted to
explore the development of pre-injury ACL damage under repetitive loading. In this study,
we ask one key question: What level of damage, if any, would the human ACL sustain
under routine mechanical loading? If the knee joint experiences 2-3 times body weight34

during normal walking10 and 3-4.5 times body weight during light sport activities such as
jogging, golf and tennis.11, 12 In previous studies, Wojtys et al have demonstrated in vitro
that the ACL could experience fatigue failure in fewer than 100 repeated landings withlimited internal femoral rotation.13 The characterization of ACL damage from thoseloading tests is the major focus of this paper.
In this study, the ACL microdamage produced in cadaveric knees was visualized
and evaluated at multiple levels from molecular, fibril, and tissue levels. Molecular level
ACL damage was investigated using both IR spectroscopy and fluorescent collagenhybridizing peptide (CHP) probes. In the IR spectroscopy, an amide I peak shift from 1664
cm-1 to 1740 cm-1 is closely associated with ligament collagen damage. The TAMRA dye
conjugated fluorescent CHP probe can detect the molecular level unwinding of collagenafter mechanical testing.14 Fibril level collagen damage was investigated using atomic
force microscopy (AFM). Fibril voids at nanometer scale ranging from 10 nm to 100 nm
were observed after mechanical testing. Tissue level collagen damage was studied using
second harmonic generation. The mechanically tested tissue demonstrated larger fibril
voids compared to the untested control. Only one knee tested in this study led to complete
mechanical failure after loading but they all demonstrated different levels of microdamage
across all scales.
The combination of these techniques allowed systematic investigation of the ACL
microdamage under repeated loading. This microdamage helps deepen the understandingof process of pre-fracture ACL fatigue. Eventually this understanding would help guidethe development, preventive training and treatment plans for athletes and active people.
Experimental Section
35

ACL sample preparation
The cadaveric knees are tested on a custom-built apparatus to simulate repeatedsingle-leg pivot landings as described in previous report.15 One knee from each pair was
set aside as an internal control. After mechanical testing, the ACL explant was extracted
and cut in half longitudinally. Each half was embedded in a water-soluble mounting
medium SCEM (Super Cryo Embedding Medium, Section Lab). The embedded tissues(both tested and control) were sectioned using a tungsten carbide blade (Leica, #TC-65)
under -25 ℃ (Leica cryostat model 3050S, 8°, 20 μm).

The sectioned tissue wastransferred to adhesive tapes using the Kowamoto method.16, 17 The sectioned sample slices
were stored in a moisturized chamber under -20℃ until being imaged.
AFM-IR data collection
The sectioned sample slices were immersed in deionized water to remove theembedding media and keep the sample hydrolyzed. The tape containing the ACL enthesis
was cut to a ~2cm square and glued to a stainless-steel AFM sample puck. The AFM-IR
data was collected using a NanoIR2 system (Anasys Instruments). For a typical IR map,the images are taken at 2*2 μm2, co-average 256x, power 2.07% and trace speed 0.1Hz.
SHG and fluorescence data collection
The samples are stained with a CHP-TAMRA dye after the AFM-IR imaging. The
peptide sequence can be found from the previous publication.14 The peptide wassynthesized by Pierce Custom Peptides. The TAMRA fluorophore was picked so thefluorescence is not overlapping with the sample autofluorescence or the SHG signal.
Before peptide staining, 25 μL CHP stock solution (150 μM) was heated under 80 ℃ for36

10min to deaggregate the peptide. The solution was quenched by immersion in 4 ℃ waterfor 15 seconds. The aliquot was diluted to a total volume of 500 μL and dropped onto the
ACL slice. The sample was incubated in dark in a 4 ℃ fridge overnight. The sample wasrinsed with 1mL PBS three times before immersed in oil (Leica type F) for microscopic
imaging. The images are collected using a Leica SP8 confocal microscopy system. The
light source was a 910nm IR laser (10% LP, 33% gain, 38% offset, pinhole wide open).
Two PMT detectors are set at 440nm for the detection of SHG forward/backward signal.
The fluorescence signal was detected at a 570-590 nm window.
Results and Discussion
Fibril damage detected by AFM-IR37

Atomic force microscopy is a common tool of studying the collagen order andFigure. 3.1. AFM height
images of voids inligament associated withmechanically induced
microdamage. A) Failed
patient ACL, B)
Mechanically tested
cadaver ACL.

morphology at nano meter scale. The AFM images from thefailed patient ACL and the mechanically tested cadaveric ACL
were compared (figure 3.1). This data showed fibril voids
ranging from 20 nm to 150 nm on both types of tissues. The
fibril voids appear as the dark spheres on the AFM images. The
fibril voids sizes are very similar between the case of the failed
patient ACL and the mechanically tested cadaver ACL. These
sphere patterns were not observed on the untested cadaveric
control. The size range of those fibril voids falls into the size
range for collagen fibrils. It is possible that individual collagen
fibril on the ligament was pulled out during the mechanicaltesting and subsequently leaving the fibril voids on theligament.

38

Figure. 3.2. AFM-IR spectra from theligament of A) explant frommechanically tested cadaveric ACL, B)
explant from patient having failed
ACL, C) explanted of untested
cadaveric ACL.

Additionally, on the AFM-IR system, anenhanced 1740 cm-1 peak was detected on the
spectrum of the mechanically tested sample (figure3.2). This peak is shifted from the collagen amide I
peak at 1660 cm-1 and this 1740 cm-1 peak can also
be reversibly created by dehydrating/rehydrating thecollagen tissue. This disordered collagen peak canalso be observed on the fractured patient ACL andis not present on the untested cadaveric ACL control.
This IR peak demonstrated the microdamage on the
ACL could be deeper into the molecular level and at
a scale even smaller than the fibril level damage asdetected by the AFM technique. This peak is indicating the unwinding of the collagen fibril
is happening when the ACL was mechanically tested under submaximal loading.
Untested

3x body weight, 100 cycles

Figure 3.3. fibril void on ligament detected by SHG.
39

Fibril damage detected by SHG and confocal microscopy
Collagen molecules are well aligned biopolymers and can be specifically detectedby second harmonic generation. SHG imaging showed fibril voids at the micrometer scale(figure 3.3). The tested ACL demonstrated the spherical fibril voids. Fibril void analysis
on the sectioned ACL showed that the collagen SHG fibril voids on the tested ACL isapproximately ten times larger than the control ACL (figure 3.3 right). Additionally, thesamples are incubated with TAMRA conjugated collagen hybrid peptide (CHP) to detect
the molecular level damage. The CHP has a complementary sequence to the collagen andcan bind specifically to the unwound collagen molecules. The conjugated TAMRA dye on
the peptide has a red fluorescence that is away from the autofluorescence and the TAMRA
intensity could be the indication of molecular level collagen damage. The tested ACL
sample demonstrated an increased TAMRA fluorescence hence higher CHP binding
Mean Intensity (100*100um)

14-0107 LF (untested)

64yr male
14-0107 RF (tested)

14-0063 RL (untested)

43yr male
14-0063 LF (tested)

14-0052 RF (untested)

57yr female
14-0052 LF (Tested)

0

50

100

150

Figure 3.4. collagen disordering detected by CHP-TAMRA.

40

200

250

300

towards the ACL (figure 3.4). This is consistent with the IR data demonstrating themolecular level damage of the ACL after the mechanical testing.
Conclusion
This study demonstrated the evidence of the hierarchical ACL damages at different scales
after the mechanical testing. The fibril and fiber voids observed on AFM and SHG
resembles the voids observed on fractured ACL. These results support the hypothesis thatthe microdamages can result from normal mechanical loading and eventually become arisk factor for ACL injuries. More cadaveric data and the comparison between the cadaver
and patient could help develop further understanding of the non-contact ACL damages.

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Conclusions and Future Directions
The substrate-triggered polymer binding to an FBP exosite provides a new protein
binding strategy. The polymer scaffold can not only act as the drug delivery vector, butalso the booster for protein binding affinities. This design strategy may not be limited tothe design to FBP binders. This strategy could save a lot resources in the new drug design
and be resistant to single site protein mutation. The protein surface and polymer surface
interaction may be the most important factor to consider in such design. The FBP triggered
nanoparticle agglomeration could pose a risk factor in the targeted drug development. How
these nanoparticles interact with other serum proteins, and the lipid in the blood serum,
would be the next question to answer. These works could help further understand theimplications of this agglomeration for biological trafficking and potential health risks.
For the ACL project, the evidence has been accumulating that the microdamage to
the ACL occurs after the mechanical loading at the ACL without the presence of ACL
fracture. The next step is to understand how the ACL fractures develop overtime whenpeople age and ACL become repetitively loaded. A diagnostic tool, such as a SHG or
fluorescence endoscope, can be developed to help the clinicians to better illustrate thedevelopment of the ACL injury. These tools could also help the improvement of exercisingand training practices to better protect the ACL of athletes or common people.

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