applied
sciences
Review
Collagen Nanoparticles in Drug Delivery Systems and
Tissue Engineering
Ashni Arun 1,† , Pratyusha Malrautu 1,† , Anindita Laha 1, *, Hongrong Luo 2, *






Citation: Arun, A.; Malrautu, P.;
Laha, A.; Luo, H.; Ramakrishna, S.
Collagen Nanoparticles in Drug
Delivery Systems and Tissue
Engineering. Appl. Sci. 2021, 11,
11369. https://doi.org/10.3390/
app112311369
Academic Editor: Suwan N.
Jayasinghe
Received: 11 August 2021
Accepted: 16 October 2021
Published: 1 December 2021
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
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Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Appl. Sci. 2021, 11, 11369. https://doi.org/10.3390/app112311369
and Seeram Ramakrishna 3
1 Department of Chemical Engineering, Manipal Institute of Technology, Manipal Academy of Higher
Education, Manipal 576104, India; ashniarun2018@gmail.com (A.A.);
pratyusha.malrautu99@gmail.com (P.M.)
2 Engineering Research Center in Biomaterials, Sichuan University, Chengdu 610064, China
3 Center of Nanofibers and Nanotechnology, National University of Singapore, Singapore 117581, Singapore;
seeram@nus.edu.sg
* Correspondence: anindita.laha19@gmail.com (A.L.); hluo@scu.edu.cn (H.L.)
† Both authors contributed equally.
Abstract: The versatile natural polymer, collagen, has gained vast attention in biomedicine. Due to
its biocompatibility, biodegradability, weak antigenicity, biomimetics and well-known safety profile,
it is widely used as a drug, protein and gene carrier, and as a scaffold matrix in tissue engineering.
Nanoparticles develop favorable chemical and physical properties such as increased drug half-life,
improved hydrophobic drug solubility and controlled and targeted drug release. Their reduced
toxicity, controllable characteristics of scaffolds and stimuli-responsive behavior make them suitable
in regenerative medicine and tissue engineering. Collagen associates and absorbs nanoparticles
leading to significant impacts on their biological functioning in any biofluid. This review will discuss
collagen nanoparticle preparation methods and their applications and developments in drug delivery
systems and tissue engineering.
Keywords: drug delivery systems (DDS); tissue engineering (TE); polymers; collagen; nanoparticle (NP)
1. Introduction
Collagen is an abundant protein found in the human body existing in bones, tendons,
muscles and skin [1]. Properties such as tensile strength, biodegradability, notable stretcha-
bility, absorption in-vivo capacity, biocompatibility, biomimetic nature, weak antigenicity,
and remarkable safety profile make it a significant element in applications of drug delivery
systems (DDS) and tissue engineering (TE) [2]. Collagen has good in vivo absorption, syn-
ergism with bioactive materials, haemostatic characteristics, and low immunogenicity [3,4].
To overcome limitations such as enzymatic degradation, weak mechanical strength and
low thermal stability, methods such as crosslinking, grafting polymerization, blending
and covalent conjugation are adopted, resulting in biomaterials of discrete physical forms
such as shields, films, sponges, hydrogels, microspheres, sheets, coatings, liposomes, disks,
nanofibers, tablets, pellets and nanoparticles (NPs) [5,6].
Collagen acts as an efficient carrier for the delivery of various agents such as genes,
drugs, proteins and growth factors. It is possible to alter collagen to fabricate materials of
various durabilities, structures and forms due to its adaptable nature. Collagen can also
form complexes with different biologically active and medical substances [7]. Some of
the applications of collagen are the formation of microspheres and microneedles for drug
delivery [8], formulation of NPs for gene delivery [9], development of pellets and tablets
for protein delivery [10], formation of gels and combination with liposomes for sustained
drug delivery [11], cancer treatment [12] and collagen shields in ophthalmology [13]. The
collagen types that can be purified and isolated for usage in pharmaceutical industries for
drug delivery are: (i) Enzyme and alkali treated collagen, (ii) natural salt-soluble collagen,
https://www.mdpi.com/journal/applsci
Appl. Sci. 2021, 11, 11369 2 of 17

(iii) insoluble collagen and (iv) acid-soluble collagen [14]. The degradation of collagen-
based biomaterials for TE applications could possibly lead to tissue functionality and
structure restoration. A few applications of collagen in TE have been utilised in cornea
and skin related treatments [15–17], osteochondral defects [18], wound dressing, dermal
filler and delivery systems [17]. Collagen-based wound dressings are used for applications
of wound and burn coverage, ulcer treatment, therapeutic enzyme immobilization, an-
tithrombogenic surfaces and bone filling substances [3]. When combined with elastic which
provides flexibility, collagen supports and provides firmness and strength to body tissues
and organs [18]. The usage of collagen as skin substitutes led to its usage in bioengineering
of tissues such as ligament and blood vessels [19].
Particles with diameters 1–100 nm are called NPs. DDS based on NPs display en-
hanced efficacy of drugs and improve the drug’s half-life, hydrophobic drug solubility
and controlled and sustained drug release in the infected regions. Stimuli-responsive
NPs regulate drug biodistribution and reduce drug toxicity [20,21]. TE requires the local
controlled delivery of these bioactive and contrast compounds to deploy control over
and monitor the tissues that are engineered. This need of TE is satisfied by NPs as they
control scaffold characteristics such as mechanical strength and yield regulated release
of bioactive compounds [22,23]. NPs overcome limitations such as unstable bioactivity,
reduced half-life of bioactive and contrast compounds and low solubility, which makes
them favourable for bioactive compound delivery and monitoring applications such as
specific site imaging, cell patterning, biosensing, DNA structure probing and detection of
molecules [23–25].
Polymeric NPs possess reduced cytotoxicity, high permeation and retention (EPR)
effect, good biocompatibility and are able to deliver drugs which are poorly soluble and
also control their release. They retain bioactivity of bioactive compounds from degradation
of enzymes which makes them suitable to overcome problems in tissue engineering and
regenerative medicine (TERM) applications [20,26]. Current PNPs systems are sensitive
to stimuli such as temperature, light, pH, oxidizing/reducing agents, magnetic fields and
enzymes which increases efficiency and specificity for TERM applications [21] king of
collagen with NPs results in stabilization of the nanoparticles and helps with entrapment
of the drug, to attain steady and regulated drug release for ideal therapeutic reactions.
Collagen associates and absorbs NPs, leading to significant impacts on their biological
functioning in any biofluid [27,28]. Collagen NPs are advantageous over other natural and
synthetic polymeric NPs as they have favourable biocompatibility and biodegradability,
low antigenicity, high contact surface, reduced toxicity and high cationic-charge density
potential as they possess many amino groups; in the absence of surface modification with
target compounds, they have a restricted capacity to cross the blood brain barrier. They
are small sized, have large surface area and absorptive capability and the ability to diffuse
in water to form colloidal solutions. Collagen NPs are also sterilized easily, thermally
stable, improve cell retention and decrease effects of toxic by-products formed during
degradation [29]. This is supported by experiments conducted by Alarcon et al. in which
silver NPs when formulated with collagen type I showed great stability. In this study,
spherical silver NPs with a diameter of 3.5 nm, stabilized in collagen, were prepared by a
photochemical method at room temperature. This nanocomposite (AgNP@collagen) based
on collagen displayed a nontoxic nature and remarkable biocompatibility and antibacterial
characteristics. When examined, it was proved that bactericidal characteristics shown by
AgNP@collagen were due to the presence of NPs [30].
This review will discuss the examples of collagen enhancing the properties of NPs,
their chemical and physical preparatory methods and various developments and applica-
tions of these collagen NPs in DDS and TE.

2. Methods of Collagen Nanoparticle Preparation

Protein nanoparticle preparation and the process of drug encapsulation are carried
out in moderate conditions, avoiding toxic organic solvents or chemicals. Collagen NPs
 Protein nanoparticle preparation and the process of drug encapsulation are carried
out in moderate conditions, avoiding toxic organic solvents or chemicals. Collagen NPs
can be fabricated using three methods: (i) chemical, (ii) physical and (iii) self assembly
The chemical methods are coacervation or colyelectrolyte complexation and emulsifica-
Appl. Sci. 2021, 11, 11369 tion; physical methods include nano spray drying and electrospraying; and self-assembly3 of 17
comprises desolvation [31]. Some additional methods which have been discussed are mill-
ing, interfacial polymerisation, phase separation and polymer chain collapse [10,32]. This
section discussesusing
can be fabricated thesethree
collagen nanoparticle
methods: preparation
(i) chemical, (ii) physicalmethods which
and (iii) self have specific
assembly.
advantages
The chemicaland limitations.
methods are coacervation or colyelectrolyte complexation and emulsifica-
tion; physical methods include nano spray drying and electrospraying; and self-assembly
2.1. Emulsification/Solvent
comprises desolvation [31].Extraction
Some additional methods which have been discussed are
milling, interfacial polymerisation, phase separation and polymer chain collapse [10,32].
The drug is dissolved in an organic protein or polymer solution and an emulsion
This section discusses these collagen nanoparticle preparation methods which have specific
system is formed by mechanical agitation. The polymer and drug are precipitated in drop-
advantages and limitations.
lets and NPs are formed by extracting the solvent by evaporation [33,34]. The double
emulsion method has been
2.1. Emulsification/Solvent used currently for greater efficiency of encapsulation [35]. In
Extraction
this method,
The drugaissurfactant
dissolved isin used in dispersion
an organic protein or forpolymer
emulsion stabilization.
solution The organic sol-
and an emulsion
vent
system is is
then taken
formed byout to conserve
mechanical the NPs
agitation. Thein an aqueous
polymer and drug buffer. The double in
are precipitated emulsion
droplets and NPs are formed by extracting the solvent by evaporation
method of nanoparticle preparation is quick and economical. A stabilizing agent and sur-[33,34]. The double
emulsionare
factant method has been
required used currently
to ensure for greater
the emulsion efficiency
is stable as itofisencapsulation [35]. In this
normally unstable thermody-
method, a surfactant is used in dispersion for emulsion stabilization. The organic solvent is
namically. They affect interactions between the drug and matrix along with the drug re-
then taken out to conserve the NPs in an aqueous buffer. The double emulsion method
lease rate [31]. More specifically, at room temperature, an aqueous phase of collagen along
of nanoparticle preparation is quick and economical. A stabilizing agent and surfactant
with water to
are required and a hydrophilic
ensure the emulsionsurfactant,
is stable as an
it isorganic
normallyphase
unstable consisting of a surfactant of
thermodynamically.
lipophilic
They affect nature and between
interactions a solventthemiscible
drug and inmatrix
water along
are mechanically
with the drugagitated, forming
release rate [31]. a ho-
mogeneous emulsion
More specifically, at roomsystem. This emulsion
temperature, an aqueouscombined drop by
phase of collagen alongdrop with
with hotand
water oil results
in collagen nanoparticle
a hydrophilic surfactant, anfabrication
organic phase [36]. An experiment
consisting conducted
of a surfactant by Sharkawy
of lipophilic nature andet al. was
a solvent
to develop miscible in water
new stable are mechanically
Pickering emulsions agitated,
that are forming a homogeneous
stabilized emulsion
with chitosan/collagen pep-
system. This emulsion combined drop by drop with hot oil results in collagen
tide NPs for possible cosmetic applications. Their study aims to understand the stability nanoparticle
fabrication [36]. An experiment conducted by Sharkawy et al. was to develop new stable
microstructure and rheological properties of the Pickering formulations produced. Since
Pickering emulsions that are stabilized with chitosan/collagen peptide NPs for possible
both the polymers utilized in nanoparticle production have skin benefits, the study inves-
cosmetic applications. Their study aims to understand the stability, microstructure and
tigates the fate
rheological of the of
properties NPs
theafter skin application
Pickering formulationsby trackingSince
produced. theirboth
skinthe
distribution
polymers follow
ing the penetration
utilized in nanoparticle of production
the emulsion have droplets. During
skin benefits, thethe
studycomplex chitosan/collagen
investigates the fate of pep-
tides nanoparticle formation, the long chitosan chains fold around
the NPs after skin application by tracking their skin distribution following the penetration the collagen peptides
and
of theform a new
emulsion phase complex
droplets. During the having
complex different propertiespeptides
chitosan/collagen from that of the individua
nanoparticle
formation, the[37].
components longThechitosan chains
process fold around the collagenExtraction
of Emulsification/Solvent peptides and hasform
beenaillustrated
new in
phase complex
Figure 1. having different properties from that of the individual components [37].
The process of Emulsification/Solvent Extraction has been illustrated in Figure 1.

Figure 1. Emulsification/Solvent Extraction—The drug is dissolved in the polymer solution and an

Figure 1. Emulsification/Solvent Extraction—The drug is dissolved in the polymer solution and an
emulsion is formed by mechanical agitation. The polymer and drug are precipitated in droplets and
emulsion is formed by mechanical agitation. The polymer and drug are precipitated in droplets and
NPs are
NPs arefabricated
fabricatedbyby
solvent extraction
solvent by evaporation.
extraction by evaporation.
2.2. Complex Coacervation/Polyelectrolyte Complexation
2.2. Complex
ComplexCoacervation/Polyelectrolyte Complexation
coacervation or polyelectrolyte complexation is a process where oppositely
charged polyelectrolytes
Complex in an aqueous
coacervation solution undergo
or polyelectrolyte a liquid−liquid
complexation phase separation
is a process where oppositely
charged polyelectrolytes in an aqueous solution undergo a liquid−liquid phaseThe
(LLPS) for the formation of a polymer-dilute and polymer-dense (coacervate) phase. separation
coacervate’s properties can be adjusted by the chirality of the polyelectrolytes, salt
(LLPS) for the formation of a polymer-dilute and polymer-dense (coacervate) phase. The concen-
tration, pH, charge density, temperature and ionic strength, enabling the understanding
of LLPS from changes in the physical environment [38]. It is a chemical nanoparticle
preparation method and involves the incorporation of natural salt or alcohol to a solution
of collagen which changes the collagen structure, to which a crosslinking material is added,
leading to nanoparticle formation, as explained in Figure 2. This preparation method is
 ing of LLPS from changes in the physical environment [38]. It is a chemical nanopar
preparation method and involves the incorporation of natural salt or alcohol to a solu
of collagen which changes the collagen structure, to which a crosslinking materi
added, leading to nanoparticle formation, as explained in Figure 2. This prepara
Appl. Sci. 2021, 11, 11369 4 of 17
method is comparable to the desolvation method, only differing in parameters suc
pH, rate of addition of solvent, temperature, homogenizer speed, crosslinking agent
centration, molar ratios of protein and organic solvent [36]. Due to the amphoteric na
comparable to the desolvation method, only differing in parameters such as pH, rate of
of proteins which consist of multiple charged functional groups, they can be change
addition of solvent, temperature, homogenizer speed, crosslinking agent concentration,
eitherratios
molar anionic or cationic
of protein just by
and organic modifying
solvent [36]. Due parameters such as
to the amphoteric pH.of
nature The protein whi
proteins
charged
which canof associate
consist electrostatically
multiple charged with they
functional groups, different polymeric
can be changed electrolytes
to either anionic [31].
method
or cationicisjust
useful in applications
by modifying parameters such
such asasentrapment
pH. The proteinof DNA
which for gene therapy
is charged can [39
associate electrostatically
study conducted with different
by Singh polymeric
et al. explores theelectrolytes
potential [31]. This method
of DNA is useful with n
NPs prepared
in applications such as entrapment of DNA for gene therapy [39].
collagen (NC) and methylated collagen (MC) to efficiently deliver genes into A study conducted by cells.
Singh et al. explores the potential of DNA NPs prepared with native collagen (NC) and
transfection abilities and physicochemical properties of these two types of NPs were s
methylated collagen (MC) to efficiently deliver genes into cells. The transfection abilities
ied physicochemical
and in parallel. NC/properties
DNA and ofMC/DNA NPsof
these two types were
NPs prepared
were studied using the complex
in parallel. NC/ coace
tion method.
DNA and MC/DNA It wasNPsobserved that NC
were prepared formed
using complexes
the complex with themethod.
coacervation DNA atIt awas low pH v
This complex
observed that NC accumulated
formed complexes promptlywith at
theneutral
DNA atpH andpH
a low did not give
value. out significant
This complex
accumulated
tection to DNA promptly
becauseat neutral
of its pHpoorand did not give
stability out significant
in serum. MC carriesprotection to DNA
a positive charge at
because of its poor stability in serum. MC carries a positive charge at
tral pH and thus has higher stability under physiological conditions and strong D neutral pH and thus
has higher stability under physiological conditions and strong DNA binding ability. It also
binding ability. It also showed that MC/DNA NPs were smaller and more stable
showed that MC/DNA NPs were smaller and more stable than NC/DNA particles, which
NC/DNA
released DNA particles, whichmanner
in a prolonged released forDNA
up to 3inweeks
a prolonged
[38,40]. manner for up to 3 weeks [38

Figure2.2.Complex
Figure Complex Coacervation—This
Coacervation—This method
method regulates
regulates the pH the pH to produce
to produce the proteinthecation
protein cati
anion,
or anion,and
andthen reactswith
then reacts withdifferent
different polymers
polymers to fabricate
to fabricate NPs. ItNPs. It involves
involves the incorporati
the incorporation
natural
of naturalsalt
saltor
oralcohol
alcohol to aa protein
proteinsolution
solution which
which changes
changes the protein
the protein structure,
structure, to whichto which
a ac
crosslinking material
linking material is isadded
addedleading
leading to
tocross-linked
cross-linked drug loaded
drug protein
loaded NPs. NPs.
protein
2.3. Phase Separation
2.3. Aqueous
Phase Separation
and organic phase separations are the backbone of the emulsion solvent
Aqueous
evaporation and organic
technique phaseNPs.
of fabricating separations are the
The polymers arebackbone
put down of theorganic
in an emulsion sol
solvent and the
evaporation addition of
technique ofafabricating
surfactant toNPs.
this aqueous solutionare
The polymers takes
putplace,
down to in
avoid
an organic
the
vent and the addition of a surfactant to this aqueous solution takes place, ato avoid
emulsion particle fusion [41]. This solution is further put through ultrasonication,
method for mixing. Consequently, the droplets of the polymer are formed and the solvent
emulsion particle fusion [41]. This solution is further put through ultrasonicatio
is isolated. This is often concluded by the organic phase evaporation. The leftover solution
methodoffor
consists themixing.
polymericConsequently, the dropletswith
NPs that are accumulated of the
the polymer are formed
help of a centrifuge. and the sol
Phase
is isolated.fabricates
separation This is often concluded
particles by range
in the size the organic phase
of 50–500 nm,evaporation. The leftover
which is modulated by solu
consists of the polymeric NPs that are accumulated with the help of a centrifuge. P
changing the polymer solution concentration [42]. This method is well-known because of
the conjugated
separation polymer availability
fabricates particles which
in theis size
demonstrated
range ofin50–500
the experiments
nm, whichcarried
is out
modulate
by Yoon et al. where they prepared NPs of conjugated polymers from phase separation,
changing the polymer solution concentration [42]. This method is well-known becau
which resulted in lipid-assembled NPs which can be combined with many functionalities
such as inorganic/organic nanomaterials, cell specific ligands polyethylene glycols for
in vivo applications [43]. The method of coacervation also involves phase separation,
 vivo
the applications
conjugated [43]. The
polymer method which
availability of coacervation also involves
is demonstrated phase separation,
in the experiments carriedw
needs
by Yoontheetliquid solutions’
al. where separation,
they prepared NPsoneofthat containspolymers
conjugated the solvent andphase
from the other is a
separat
tein polymer.
which resulted Coacervation is caused
in lipid-assembled by thecan
NPs which disruption
be combinedof equilibrium
with many through
functionali m
Appl. Sci. 2021, 11, 11369
such as
such asinorganic/organic
salt addition. Thenanomaterials,
charges create cell
electrostatic forces which
specific ligands induce
polyethylene the form
glycols
5 of 17
fo
vivo applications
of NPs. [43].reduces
This process The method of coacervation
the protein solubility also
which involves
leads tophase
phaseseparation,
separationwh [4
shownthe
needs in liquid
Figuresolutions’
3. separation, one that contains the solvent and the other is a p
tein polymer. Coacervation is caused by the disruption of equilibrium through me
which needs the liquid solutions’ separation, one that contains the solvent and the other is
such as salt addition. The charges create electrostatic forces which induce the forma
a protein polymer. Coacervation is caused by the disruption of equilibrium through means
of NPs.
such This
as salt process
addition. reduces
The chargesthe protein
create solubility
electrostatic forceswhich
which leads
induceto phase
the separation
formation of [44
shown
NPs. inprocess
This Figurereduces
3. the protein solubility which leads to phase separation [44] as
shown in Figure 3.

Figure 3. Phase Separation—Polymers are placed in an organic solvent and the addition of a su
tant to this aqueous solution takes place, to avoid the emulsion particle fusion. This solution i
ther put through ultrasonication, a method for mixing. Polymer droplets are formed and the so
is isolated, concluded by the organic phase evaporation.
Figure Phase Separation—Polymers
Figure3.3. Phase Separation—Polymers areare
placed in an
placed in organic solvent
an organic and and
solvent the addition of a of a sur
the addition
2.4. to
tant Nano
this Spray
surfactant to this
aqueous Drying
aqueous solution takes place, to avoid the emulsion particle fusion. This solution
solution takes place, to avoid the emulsion particle fusion. This solution is
is further put through ultrasonication, a method for mixing. Polymer droplets are formed and the
ther put through
Nano sprayultrasonication, a method nanoparticle
drying is a physical for mixing. Polymer dropletsmethod
preparation are formed and the
which sol
is use
solvent is isolated, concluded by the organic phase evaporation.
isspherical
isolated, collagen
concludednanoparticle
by the organicfabrication
phase evaporation.
in liquids. A dilute collagen solution is spr
2.4. Nano Spray Drying
into chambers at an increased temperature where hot carbon dioxide and nitrogen
2.4. Nano
Nano Spraydrying
spray Drying a physical nanoparticle preparation method which is used for
flows in the directionisof the spray from the nozzle producing hollow nanospheres w
spherical
Nano collagen
spray nanoparticle
drying is afabrication
physical innanoparticle
liquids. A dilute collagen solution
preparation is sprayed
are collected by an electrode at the bottom, as demonstrated in method
Figure 4which is usedn
[45]. Liquid
into chambers
spherical at
collagenan increased temperature where hot carbon dioxide and nitrogen gas
gen is used whennanoparticle
collagen solutionfabrication in liquids.
is sprayed A dilute denaturation
to prevent collagen solution
flows in the direction of the spray from the nozzle producing hollow nanospheres which are
whichis spra
ma
into
causedchambers
by high attemperatures.
an increased temperature
Prepared where NPs
collagen hot carbon
are then dioxide
frozen, and nitrogen
their hardne
collected by an electrode at the bottom, as demonstrated in Figure 4 [45]. Liquid nitrogen
flows
is usedin
improved whenthebydirection
tempering
collagen of the
solution andisspray
theyfrom
sprayed are the nozzle
lyophilized,
to prevent producing
crosslinked
denaturation whichhollow
withbe
may nanospheres
specific
caused agents wh
are
by collected
high
sanitized by This
temperatures.
[10]. an electrode
Prepared
is a rapid at the
andbottom,
collagen NPs are as
inexpensivethendemonstrated
frozen,
method in Figure
theirtohardness
produce 4small-scale
[45]. Liquidcoll
is improved ni
gen
by
NPs. isHydrophilic
used and
tempering when collagen
theydrugs cansolution
are lyophilized, is sprayed
crosslinked
be encapsulated with to prevent
specific
in spray agents
dried denaturation
and [46].
NPs sanitized
Spraywhich mayi
[10].drying
This
causedis a rapid
by and temperatures.
high inexpensive method to produce
Prepared small-scale
collagen NPs collagen
are then NPs. Hydrophilic
frozen, their hardnes
vantageous as the particle size can be regulated by varying parameters, such as no
drugs
improved can beby encapsulated
tempering inand
spray dried
they NPs
are [46]. Spray drying
lyophilized, is advantageous
crosslinked with specificas the agents
size and
particle sizespray
can berate. For protein
regulated by varying NPs, surfactants
parameters, arenozzle
such as added sizetoand
ensure
spray stabilization
rate. For of
sanitized
ticles of [10]. This
thesurfactants
polymerareis a rapid
[31,45]. and inexpensive method to produce small-scale colla
protein NPs, added to ensure stabilization of particles of the polymer [31,45].
NPs. Hydrophilic drugs can be encapsulated in spray dried NPs [46]. Spray drying is
vantageous as the particle size can be regulated by varying parameters, such as no
size and spray rate. For protein NPs, surfactants are added to ensure stabilization of p
ticles of the polymer [31,45].

Figure4. 4.
Figure Nano
Nano Spray
Spray Drying—A
Drying—A liquidliquid jet stream
jet stream of theisprotein
of the protein released is released
into into
a chamber a chamber
using a us
nozzleand
nozzle and dried,
dried, andand heated
heated carboncarbon
dioxidedioxide and nitrogen
and nitrogen and hollowand hollow nanospheres
nanospheres are collect
are collected by
anelectrode
an electrode at the
at the bottom
bottom of theofchamber.
the chamber.
2.5. Electrospraying
Figure 4. Nano Spray Drying—A liquid jet stream of the protein is released into a chamber usi
nozzleAsand
shown
dried,in and
Figure 5, a carbon
heated high voltage
dioxideisand
applied to a and
nitrogen solution of nanospheres
hollow protein and the
are collecte
nozzle sprays a liquid jet stream resulting
an electrode at the bottom of the chamber. in the formation of an aerosolized droplet which
consists of protein NPs in the colloidal size [47,48]. Drugs are easily able to be incorporated
 2.5. Electrospraying
As shown in Figure 5, a high voltage is applied to a solution of protein and the no
sprays a liquid jet stream resulting in the formation of an aerosolized droplet which c
Appl. Sci. 2021, 11, 11369 sists of protein NPs in the colloidal size [47,48]. Drugs are easily able to be 6 of incorpora
17
into these NPs using this process [49]. Depending on the DDS type, parameters such
voltage applied, operating distance, gauge diameter of the needle and flow rate vary.
increased voltage
into these NPs is this
using applied
processto [49].
the solution
Depending of on
thethe
polymer to make
DDS type, sure such
parameters it comes ou
the syringe as NPs [50]. The electrospraying method is economical,
as voltage applied, operating distance, gauge diameter of the needle and flow rate vary. easy to carry out
has good efficiency
An increased voltage isofapplied
encapsulation. Stable
to the solution NPs
of the can betofabricated
polymer make sure itwithout
comes out problems
of the syringe as NPs [50]. The electrospraying method is economical,
garding biocompatibility and lowered encapsulation efficiency. The newer method of easy to carry out
and has
axial good efficiency of
electrospraying encapsulation.
consists Stablespray
of a coaxial NPs can be fabricated
head so both without
solutions problems
can be guide
regarding biocompatibility and lowered encapsulation efficiency. The newer method of
the electric field [31]. In experiments conducted by Nagarajan et al, solid collagen N
coaxial electrospraying consists of a coaxial spray head so both solutions can be guided
were
to the fabricated
electric fieldin a single
[31]. step under
In experiments ambient
conducted pressure etand
by Nagarajan temperature
al, solid collagen NPs conditions
electrospray
were fabricated deposition. Electrospraying
in a single step under ambientofpressure
the collagen solution, conditions
and temperature increasingbythe solu
conductivity then using
electrospray deposition. salts to induce
Electrospraying structural
of the perturbation
collagen solution, of the
increasing thecollagen
solution molec
conductivity then using salts to induce structural perturbation of the collagen
formed solid NPs. These solid collagen particles had high potential to act as drug carr molecules
formed solid NPs. These solid collagen particles had high potential to act as drug carriers
and this was shown by utilizing theophylline as a model drug using the coaxial sp
and this was shown by utilizing theophylline as a model drug using the coaxial spray
technique. Release of theophylline was regulated by crosslinking of the molecules of
technique. Release of theophylline was regulated by crosslinking of the molecules of
lagen. This
collagen. proved
This provedthat
that electrospray deposition
electrospray deposition waswas a favourable
a favourable preparatory
preparatory methodmethod
fabrication of of
for fabrication solid
solidcollagen NPstotobebeused
collagen NPs used in drug
in drug delivery
delivery applications
applications [51]. [51].

Figure 5. Electrospraying—NPs
Figure 5. Electrospraying—NPs areare fabricated
fabricated by releasing
by releasing a liquid
a liquid jet stream
jet stream throughthrough
a nozzlea nozzle
(coaxial
axial needle),forming
needle), forming ananaerosolized
aerosolizeddroplet afterafter
droplet exerting high voltage
exerting high to the protein
voltage solution.
to the protein solut
High voltage is applied to the polymer solution to make sure it comes out of the syringe
High voltage is applied to the polymer solution to make sure it comes out of the syringe as NP as NPs.

2.6. Self Assembly

2.6. Self Assembly
Proteins that are modified hydrophobically, when added to aqueous solutions, can
Proteins thattoare
be self-assembled formmodified hydrophobically,
micelle NPs. These hydrophobic whencores
addedcan tobe aqueous
a channel solutions,
for
active molecules. In this method of self-assembly, chains of individual
be self-assembled to form micelle NPs. These hydrophobic cores can be a channel for proteins are dis-
solved
tive in solution
molecules. Inwhich exceeds of
this method theself-assembly,
critical micelle chains
concentration, at a critical
of individual solution
proteins are dissol
temperature to enable the formation of nanoscale aggregates [52]. Upon the formation of
in solution which exceeds the critical micelle concentration, at a critical solution temp
a bridge between the chains, these micelles can be made steady through the process of
ture to enableAthe
solidification. formation
number of nanoscale
of studies aggregates
have reported [52]. Uponwound
that collagen-based the formation
dressings of a bri
between
engineered the
viachains, thesehave
self-assembly micelles can be made
been demonstrated to steady
promotethrough
fibroblast the process
production andof solidif
tion. A number
accelerate woundof studies
healing have
[53]. Thereported
controlledthat collagen-based
release of biomolecules wound dressings enginee
in collagen-based
bioactive
via wound dressings
self-assembly have been can demonstrated
be achieved by varying the self-assembly
to promote conditions ofand acce
fibroblast production
thewound
ate collagenhealing
constructs [27,54].
[53]. Vedanayagam
The controlled et al. of
release prepared varied sizes
biomolecules of silver NPs bioac
in collagen-based
(AgNPs)—10 nm, 35 nm, and 55 nm—using nutraceuticals such as Pectin as stabilization
wound dressings can be achieved by varying the self-assembly conditions of the colla
and reducing agents through the method of microwave irradiation. These AgPectin NPs
constructs [27,54].
were accumulated Vedanayagam
in the et al. prepared
self-assembly process of collagenvaried
which ledsizes of silverCollagen-
to fabricated NPs (AgNPs)—
nm, 35 nm,
Ag-Pectin and 55 nm—using
nanoparticle-based nutraceuticals
scaffolds. The in-vitro such as Pectin as analysis
biocompatibility stabilization
revealedand reduc
agents
that thethrough the method
collagen-Ag-Pectin of microwave
NP-based irradiation.
scaffold showed These
greater AgPectin
antibacterial NPs and
activity were accum
lated in the self-assembly process of collagen which led to fabricated Collagen-Ag-Pe
 nanoparticle-based scaffolds. The in-vitro biocompatibility analysis revealed that
lagen-Ag-Pectin NP-based scaffold showed greater antibacterial activity and im
Appl. Sci. 2021, 11, 11369 cell viability towards keratinocytes. Their study opened up the potential
7 of 17of utiliz
pectin caged silver NPs to develop collagen-based nanoconstructs for various biom
applications such as drug delivery and TE.
improved cell viability towards keratinocytes. Their study opened up the potential of
2.7. Desolvation
utilizing the pectin caged silver NPs to develop collagen-based nanoconstructs for various
biomedical applications such as drug delivery and TE.
Desolvation or simple coacervation is a self-assembly method which involves
corporation
2.7. Desolvation of a desolvation factor such as a natural salt or alcohol to a collagen s
containing a drug.
Desolvation Thiscoacervation
or simple desolvationis factor changesmethod
a self-assembly collagen’s
whichstructure and decre
involves the
solubility. After
incorporation a critical factor
of a desolvation amount suchof
as desolvation,
a natural salt oraalcohol
crosslinking agent
to a collagen such as glu
solution
containing a drug. This desolvation factor changes collagen’s structure
hyde is added to the formed mass of collagen which results in nanoparticle and decreases its sol-formati
ubility. After a critical amount of desolvation, a crosslinking agent such as glutaraldehyde
It is widely used in the fabrication of protein NPs [55]. Protein-based NPs which
is added to the formed mass of collagen which results in nanoparticle formation [36]. It is
ricatedused
widely by in
desolvation can
the fabrication of alter
proteinthe
NPssize
[55].ofProtein-based
particles relative to conditions
NPs which are fabricatedsuch as
tration
by of thecan
desolvation protein,
alter thepH,
sizeadditive speed
of particles relativeoftothe desolvating
conditions such asfactor and temperatu
concentration
creased
of concentration
the protein, pH, additiveofspeed
the protein and increased
of the desolvating pHtemperature.
factor and produces NPs which are sm
Decreased
concentration
size. Concentration of the protein takes place by decreasing the proteinsize.
of the protein and increased pH produces NPs which are smaller in solubility
Concentration of the protein takes place by decreasing the protein solubility by addition of
dition of the desolvating factor [31]. This process has been illustrated in Figure 6.
the desolvating factor [31]. This process has been illustrated in Figure 6.

Figure 6. Self-assembly—Desolvation—Individual protein chains are dissolved in a solution at a

Figure 6. Self-assembly—Desolvation—Individual protein chains are dissolved in a solu
Critical Solution Temperature which exceeds the Critical Micelle Concentration and leads to the
Critical Solution Temperature which exceeds the Critical Micelle Concentration and lead
generation of protein micelles during the nanosized aggregate formation, in the self-assembly method.
generation of protein micelles during the nanosized aggregate formation, in the self-a
In desolvation, NPs are fabricated by the addition of a desolvating agent to a protein solution which
method.
contains In desolvation, NPs are fabricated by the addition of a desolvating agent to a prot
drugs.
tion which contains drugs.
2.8. Milling
2.8. Collagen
Milling in the nano-scale can be produced through the process of milling. In this
process, a polymer material is disintegrated into finer NPs by the application of mechanical
energy. Collagen in the
This method nano-scale
uses canforbecarrying
milling balls produced through
out the the process
high energy mechanicalof milling
process, a polymer material is disintegrated into finer NPs by the
collision for the polymer disintegration (Figure 7). It is an economical method to carry out application of m
the
icalreduction
energy.ofThis the size of the particle
method on a large
uses milling scalefor
balls of production
carrying out [56].the
Heat energy
high in
energy mec
the process is produced as a result of the kinetic and mechanical energy that exists in the
collision for the polymer disintegration (Figure 7). It is an economical method to ca
vessel used for milling [57]. Due to this production of heat energy, the milling vessel has to
thecooled
be reduction
in orderoftothe size
avoid of the particle
degradation on a large
of the material scale of production
or overheating. [56].aHeat en
Collagen, being
the process
material is produced
sensitive as aisresult
to temperature, milledof the kineticutilising
mechanically and mechanical
liquid nitrogen energy that exist
to avoid
vessel used for milling [57]. Due to this production of heat energy, the milling ve
denaturation from heat, at cryogenic temperatures (below approximately − 150 degree
Celsius) [32]. Experiments
to be cooled in order towere conducted
avoid to fabricate
degradation small-sized
of the material collagen NPs (averageCollagen
or overheating.
size: 200 nm) using the milling technique. Micron-scale collagen powder was collected
a material sensitive to temperature, is milled mechanically utilising liquid nitr
and added into liquid nitrogen for half an hour. The cryo collagen was put in vacuum
avoid
for denaturation
2 days fromfrom
to sublimate water heat,collagen.
at cryogenic temperatures
Freeze-dried collagen was (below approximately
then subjected to -
greeenergy
high Celsius) [32].toExperiments
milling were
prepare very fine conducted
nano-scale to fabricate
collagen small-sized
particles. For every millingcollagen N
round, the amount of freeze-dried collagen was 1:5 by weight ratio
erage size: 200 nm) using the milling technique. Micron-scale collagen powder with milling balls in a w
milling container (120 mL volume). These collagen NPs have been looked
lected and added into liquid nitrogen for half an hour. The cryo collagen was put into using cell
culture tests with osteoblasts, and resulted in 30% enhancement of growth of the cell when
uum for 2 days to sublimate water from collagen. Freeze-dried collagen was th
compared to collagen particles in the micro scale [58].
jected to high energy milling to prepare very fine nano-scale collagen particles. Fo
milling round, the amount of freeze-dried collagen was 1:5 by weight ratio with
balls in a milling container (120 mL volume). These collagen NPs have been look
using cell culture tests with osteoblasts, and resulted in 30% enhancement of gro
the cell when compared to collagen particles in the micro scale [58].
Appl. Sci. 2021, 11, x FOR PEER REVIEW

Appl. Sci. 2021, 11, 11369 8 of 17

Appl. Sci. 2021, 11, x FOR PEER REVIEW 8 of 19

Figure 7. Milling—A polymer material is broken down into finer NPs by the application of me
ical energy by the rotation of a milling bowl. There are milling balls for performing high e
mechanical collisions for the polymer breakdown, using centrifugal force.
Figure 7.
Figure 7. Milling—A
Milling—A polymer
polymer material
material is
is broken
broken down
down into
into finer
finer NPs
NPs by
by the
the application
application of mechan-
mechan-
ical energy
ical energy by
by the
the rotation
rotation of
of aamilling
millingbowl.
bowl. There
There are
are milling
milling balls
balls for
for performing
performing high
high energy
energy
2.9. Interfacial
mechanical
mechanical Polymerization
collisions
collisions for the
for the polymer
polymer breakdown,
breakdown,using
usingcentrifugal
centrifugalforce.
force.
Interfacial polymerization is a method used to produce polymer membranes and
2.9.
2.9. Interfacial
Interfacial Polymerization
Polymerization
ticles. It is a polymerisation technique which takes place between the two immis
Interfacial
Interfacial polymerization
polymerizationis is aa method
method used
used to produce polymer
polymer membranes
membranes and par-
phase
ticles.
interfaces and results in the formation of a polymer which is strained to the inte
ticles. ItItisisa apolymerisation
polymerisation technique
technique which takes
which placeplace
takes between the two
between theimmiscible phase
two immiscible
[59]. In
interfaces
phase this
and process,
interfaces results an interfacial
in the
and results formation ofpolymer
in the formation a polymer iswhich
developed
of a polymer isat
is strained
which the protein
to the
strained expression
tointerface [59].
the interface sit
Inlowing
this
[59]. theprocess,
Inprocess,
this polymer straining
an interfacial polymer
an interfacial withisadeveloped
polymer suitable dye.
is developedat theItprotein
at provides
the these
expression
protein polymer
expressionsite follow-materials
site fol-
ing the polymer
distinctive
lowing the polymer straining
chemical with
and
straining a suitable
topological
with dye.
a suitable It It
provides
providesthese
characteristics
dye. thesepolymer
such as materials
alternative
polymer materials with
surface
with chem
distinctive
distinctive chemical
chemical and
hollow structures or topological
and topological characteristics
anisotropic shapes. Thesuch
characteristics such as
as alternative
materialsalternative surface
surface chemistry,
synthesized chemistry,
can be classified
hollow
hollow structures
structures or
or anisotropic
anisotropic shapes.
shapes. The
The materials
materials synthesized
synthesized can
can be
be classified
classified into
into [60].
zero-dimensional NPs, 2D films, 3D composite membranes and 1D nanofibers
zero-dimensional NPs, 2D films, 3D composite membranes and
zero-dimensional NPs, 2D films, 3D composite membranes and 1D nanofibers [60]. This 1D nanofibers [60]. This
process is
process explainedFigure
in Figure 8.
process is is explained
explainedin in Figure8.8.

Figure 8. Interfacial polymerization—It occurs between the two immiscible phase interfaces, and
results
Figure
Figure in8.
8. formation
Interfacial of apolymerization—It
Interfacial polymer strained
polymerization—It to the
occurs interface.
occurs
between theAn
twointerfacial
between the twopolymer
immiscible is developed
immiscible phase
phase interfaces, at
interfaces
and
the protein
results in expression
formation of site
a following
polymer the
strainedpolymer
to the straining
interface. with
An a suitable
interfacial dye. The
polymer
results in formation of a polymer strained to the interface. An interfacial polymer is develop figure
is shows
developed
formation
at theproteinof a expression
protein prepolymer upon the addition of polymer and oil, with
and further mixing
dye.and polymer-
the expressionsite sitefollowing
following thethe
polymer
polymer straining
straining witha suitable
a suitable The
dye.figure
The figure s
isation leads to the formation of a capsule with polymer wall.
shows formation
formation of a prepolymer
of a prepolymer upon
upon theaddition
the additionof of polymer
polymer and andoil,
oil,and
and further
furthermixing
mixingand and poly
polymerisation leads to the formation of a capsule with
isation leads to the formation of a capsule with polymer wall. polymer wall.
2.10. Polymer Chain Collapse
2.10. Single-chain
Polymer ChainpolymerCollapse NPs (SCNP) are fabricated using the polymer chain collapse
2.10.
method, Polymer
which can
Single-chain Chain
polymerCollapse
fabricate particles
NPs (SCNP) with
are great stability
fabricated in the
using the 5–20 nm range
polymer chain [61]. Dis-
collapse
method, Single-chain
tinct molecules
which can polymer
arefabricate
produced NPs
due
particles (SCNP)
towith
the control areof fabricated
great stabilitythe in
precursor
the 5–20using
chain the
nm range which polymer
[61].can chain col
direct
Distinct
molecules
the are
nanoparticle produced
propertiesdue to
[62]. the control
Different of
kinds theof precursor
the SCNP chain
method
method, which can fabricate particles with great stability in the 5–20 nm range [61] which
exist canand direct
the the
reac-
nanoparticle
tion properties [62]. Different kinds of are
the involved.
SCNP method exist and the reaction
tincttype is based
molecules on
aretheproduced
functional groups
due to that
the control of theIntramolecular
precursor crosslinking
chain which can d
type is based on the functional groups that are involved. Intramolecular
is, however, more advantageous than the intermolecular cross-linking for these methods crosslinking is,
the
however,
nanoparticle properties
more advantageous
[62].
thanchain
Different
the intermolecular
kinds of the
cross-linking
SCNP method exist and the
[63]. In homofunctional polymer collapse, a functional groupfor thesecan
which methods [63].
attach with
Intion
itself type
on theisprecursor
homofunctional based onchain
polymer thechain
functional
collapse,
is placed, groups
whichthat
a functional
after are involved.
group
a reaction which canIntramolecular
that couples attach
the with itself crosslin
functional
onis,the
grouphowever,
precursor more
is performed chain advantageous
is placed, after
(single-chain polymerthanNPs).
which the intermolecular
a reaction
However, couplescross-linking
that particles the are not for
functional
which these met
group
spher-
is performed
[63].
ical inIn shape (single-chain
are produced polymer
homofunctional using thisNPs).
polymer chain However,
method. collapse, particles which
a functional
Heterofunctional are not
group
coupling, spherical
which
which can attach
needs
in
twoshape
itself onaretheproduced
perpendicularly
precursor using this method.
cross-linked
chain placed,Heterofunctional
isfunctional groups,
after acoupling,
has therefore
which reactionbeenwhich
looked
that needs
intotwo
couples for
the funct
perpendicularly
better results. It cross-linked
is shown functional
through data groups,
that thishas therefore
method can been looked
produce NPs into
with for
a better
better
group is performed (single-chain polymer NPs). However, particles which are not sp
ical in shape are produced using this method. Heterofunctional coupling, which n
two perpendicularly cross-linked functional groups, has therefore been looked int
better results. It is shown through data that this method can produce NPs with a b
Appl. Sci. 2021, 11, x FOR PEER REVIEW
Appl. Sci. 2021, 11, 11369 9 of 17

globular shape [44,64]. Intramolecular crosslinking of a cross linkable polyme

results. It is shown through data that this method can produce NPs with a better globular
played
shape in Figure
[44,64]. 9.
Intramolecular crosslinking of a cross linkable polymer is displayed in
Figure 9.

Figure 9. Polymer Chain Collapse—Distinct molecules are fabricated due to the precursor chain
Figure 9. Polymer Chain Collapse—Distinct molecules are fabricated due to the precur
control that can direct the nanoparticle properties. Through intramolecular crosslinking, the polymer
control that can direct the nanoparticle properties. Through intramolecular crosslinking,
is fabricated to form single chain polymer NPs that find various applications in the human body.
mer is fabricated to form single chain polymer NPs that find various applications in th
body.
The advantages and limitations of these various methods of collagen NP preparation
have been discussed in Table 1.
The advantages
Table 1. Advantages andof limitations
and limitations of these methods.
collagen NP preparation various methods of collagen NP pre
have been discussed in Table 1.
Preparation Method Advantages Limitations References
Require NP
Table 1. Advantages and limitations of collagen stabilizer and surfactant
preparation methods.
Process is simple, equipment required is simple, because of unstable thermodynamic
Emulsification/Solvent
recovery can be controlled, high flexibility and nature, need to add organic solvent and [10,34]
Extraction
Preparation selectivity then remove it, residues of organic
Advantages Limitations
solvent may be toxic Refe
Method
Particles formed are very stable, NPs of smaller
Complex Coacerva-
size, by guiding process conditions, nanoparticle
tion/Polyelectrolyte Process is simple,
size and shape equipment
can be controlled, can be RequireHard
stabilizer and
process to surfactant
scale up because
[36,65] of
Complexation
Emulsification/Sol required is simple,
combined recovery
with sensitive drugscan unstable thermodynamic nature, need to
[1
vent Extraction be controlled,
Specialized highis flexibility
apparatus not necessary,and
particle addLimited
organic solvent
particle and then remove it,
size diameter,
Phase Separation size controllable by altering polymer solution small-scale production, organic solvent [43,44]
selectivity
concentration, uniform particles are formed residues ofrequirement
organic solvent may be toxic
Economical process, simple to carry out
Particles formed are very Small scale production, difficult to
experimentally, encapsulation of stable,
hydrophilic
integrate hydrophobic drugs, reduced
Nano Spray drying
Complex drugs takes place easily,
NPs of smaller size, by guiding beneficial for [45,46]
encapsulation efficiency, great energy
heat-sensitive samples as it helps maintain
Coacervation/Poly process conditions, nanoparticle consumption
temperature of the nanoparticle droplets
Hard process to scale up [3
electrolyte size
Canand shape
be scaled for can be controlled,
industrial use, good drug
Complexation loading efficiency, ease of particle
can be combined with sensitive synthesis due Reduced flow, could produce
Electrospraying [49,50]
to single-step processing, formation of dry degradation of macromolecules
drugs
particles
Highly stable process, small NPs can be formed Hard to control NP size, shape and the
Self-Assembly [27,54]
Specialized apparatusefficiency
with high encapsulation is not potential of protein strain exists

Desolvation/Simple
necessary,
Increased particle
encapsulation size Size and
efficiency, Can be carried out only for proteins
shape of NPs can be controlled using reaction Limited particle
influenced size or
by dissolution diameter,
diluted by small-scale
[31,55]
PhaseCoacervation
Separation controllable by altering polymer
conditions. carrier proteins
[4
production, organic solvent requirement
solution concentration, uniform Chamber has to be cooled due to heat
Economical, easy experimentation, controllable
Milling particles are formed
NP size, large scale prodcution
release, uncontrollable NP shape, can [57]
be carried out only for coarse NPs
Interfacial Economical Expensive polymer monomer, takes a
Easy to carry out,process,
inessentialsimple
monomerto
purity [59,60]
Polymerization lot of time to be carried out
carry out experimentally,
Polymer Chain Controllable NP properties, high stability, Limited particle diameter, hard to
encapsulation of hydrophilic [62,63]
Collapse enhanced spherical shape particle production
Smallcontrol
scaleside reaction occurrence
production, difficult to integrate
Nano Spray drugs takes place easily,
hydrophobic drugs, reduced encapsulation [4
drying beneficial for heat-sensitive
efficiency, great energy consumption
samples as it helps maintain
temperature of the nanoparticle
 Polymerization monomer purity time to be carried out

Controllable NP properties, high

Polymer Chain Limited particle diameter, hard to control
stability, enhanced spherical [62,63]
Collapse side reaction occurrence
shape particle production
Appl. Sci. 2021, 11, 11369 10 of 17

3. Collagen-Based NPs in Biomedicine

Collagen NPs NPs
3. Collagen-Based are good prospects for regulated drug release strategies as their phys-
in Biomedicine
ical characteristics such as absorption capacity, surface area and size are simple to config-
Collagen NPs are good prospects for regulated drug release strategies as their physical
ure [44,66]. They are also suitable for cell and gene delivery systems as they can defend
characteristics such as absorption capacity, surface area and size are simple to configure [44,66].
the environment of the body [67]. Temperature, pH and molecular weight of collagen are
They are also suitable for cell and gene delivery systems as they can defend the environment
determining factors for the stability of collagen NPs. Dermal delivery of retinol present in
of the body [67]. Temperature, pH and molecular weight of collagen are determining factors
collagen NPs showed that retinol was more stable due to enhanced properties of these
for the stability of collagen NPs. Dermal delivery of retinol present in collagen NPs showed
collagen NPs (Figure 10) and it displayed a faster transportation of incorporated drugs
that retinol was more stable due to enhanced properties of these collagen NPs (Figure 10)
through the skin.
and it displayed a faster transportation of incorporated drugs through the skin.

Figure 10. Enhanced properties of collagen NPs in DDS

DDS and
and TE.
TE.

The
The formation
formation ofof NPs
NPs is
is guided
guided by electrostatic forces
by electrostatic forces along
along with
with dissolving
dissolving reagents
reagents
for higher inter-charge reactions between collagen and plasmid DNA.
for higher inter-charge reactions between collagen and plasmid DNA. They can be They can be ab-
ab-
sorbed by the reticuloendothelial system and allow the amplified uptake of
sorbed by the reticuloendothelial system and allow the amplified uptake of drugs into drugs into
macrophages
macrophages andand other
other cells
cells of
of the
the body
body [68].
[68]. This
This ability
ability of
of the
the collagen
collagen NPs
NPs enables
enables them
them
to act as systemic delivery carriers for therapeutic compounds and cytotoxic
to act as systemic delivery carriers for therapeutic compounds and cytotoxic agents agents [3].
[3].
Collagen nanoparticle scaffolds can effortlessly enter wounds in the skin, which makes
them ideal for wound healing and regulated delivery of drugs [29]. Other applications
of collagen based NPs in the biomedical field include vascular, bone and skin grafting,
insertion of bio scaffolds, wound healing fillers, cartilage and nerve tissue regeneration [32].
The following section will discuss some of the notable experimental applications of collagen
NPs in drug delivery and TE.

3.1. Collagen Based NPs in DDS

Rathore et al. aimed at investigating the neuroprotective effect of the silymarin—
collagen nanoparticle DDS. They studied the importance of NPs in improving the ther-
apeutic effect of silymarin against neuronal injury. Collagen NPs were fabricated and
stabilized using malondialdehyde(MDA) and 3-ethyl carbodiimide-hydrochloride (EDC-
Hcl) as crosslinking agents. The collagen NPs that were fabricated possessed a loading
efficiency of 3.17 ± 0.37%, entrapment efficiency of 76.7 ± 2.4%, and displayed a regulated
and slow release. Their study concluded that the encapsulation of silymarin into collagen
NPs improved the therapeutic efficacy of silymarin by enhancing its bioavailability [69].
Surface modified collagen nanospheres which are delivered to the bloodstream are an-
other favorable method for drug delivery to the brain. Modification of the surface in the
spheres enables the identification of the particular surface receptors of the cell which allow
their transcytosis through modification of the sphere properties such as surface charge
and hydrophobicity, or blood-brain barrier (BBB) [70,71]. Wohlfart et al. studied that the
modification of surface in these nanospheres may allow surface receptor recognition of
a particular cell which, in turn, allows the modification of the physiological properties
of the nanospheres or the transcytosis through BBB, to help them pass over BBB through
adsorption carried out by endothelial cells [72–74].
Appl. Sci. 2021, 11, 11369 11 of 17

Mondal et al. loaded gold NPs (Au) on hydroxyapatite (HAp) surface using a rapid
microwave assisted technique. They prepared varied concentrations of Au-loaded nanos-
tructures covered with collagen (Au–HAp–Col), which were enhanced for the loading and
releasing of the doxorubicin drug, for applications in biomedicine. Collagen and Au-HAp
NPs have an electrostatic interaction between them which makes a steady nanostructure.
A ph-receptive release of approximately 53% and the highest efficiency of drug loading of
approximately 58.22% were procured for 0.1 weight percent of Au-HAp-Col NPs. Their
results demonstrated that these enhanced Au–HAp–Col NPs with bioactive and biocom-
patible characteristics could be useful for drug delivery, scaffold materials, cell growth,
proliferation and adhesion [73]. In this study conducted by Suresh et al., silver NPs (DdAg-
NPs) were synthesized with collagen and doxycycline (DO) and its strength to be utilised
as a bactericidal agent was evaluated. It was found that the DdAgNPs combined with
collagen displayed stronger antibacterial action against all the test organisms compared to
the DdAgNPs alone [74].
Another study showed that collagen NPs can be helpful in tumor infiltration for
anti-cancer drug delivery. This is due to the advantage of collagen which resembles the
microenvironment of the tumor. In this study, they developed collagen-based tumor
spheroids and optimized them using 95-D, U87 and HCT116 cells. The antitumor and
delivery efficiencies of the drug-conjugated NPs in this model were studied through
cytotoxicity and uptake studies. Their results demonstrated that the conjugated NPs reach
the tumor cells by perforating into the gel matrix. This model was found to be more
precise in figuring out the therapeutic results and dynamics of the drug transport agents
in vivo, and in the demonstration of tumor biology, which fastens the drug discovery for
cancer therapy [75]. Studies have also demonstrated that collagen NPs have been utilised
as parenteral carriers for therapeutic and cytotoxic substances, such as hydrocortisone
(Berthold et al.) and camptothecin (Yang et al.) [8,76,77] These investigations on collagen
in DDS are summarized in Table 2.

Table 2. Investigations on collagen NPs in DDS.

Agent (Crosslinking/
Stabilising/Optimising)
Malondialdehyde(MDA);
3-ethyl
carbodiimide-hydrochloride
(EDC-Hcl)
Gold NPs (Au) on
hydroxyapatite (HAp) surface
Silver NPs (DdAgNPs) with
doxycycline (DO)
95-D, U87 and HCT116 cells
Effect of Collagen NPs Application Reference
Enhanced bioavailability and improved
Drug for Neuronal injury [69]
therapeutic effect of silymarin drug
Enables identification of the particular cell
Drug delivery to the brain [70]
surface receptors which allows transcytosis
Enhancement of motor functions in PD model Nerve Growth Factor (NGF)
[73]
and cognitive functions in AD model delivered to the brain
Enhanced Au–HAp–Col NPs with bioactive and Drug delivery, scaffold
biocompatible characteristics for loading and materials, cell growth, [74]
releasing of the doxorubicin drug proliferation and adhesion
Stronger antibacterial action against all the test
Bactericidal agent [75]
organisms compared to the DdAgNPs alone
More precise therapeutic results and dynamics Tumor infiltration for
[77]
of the drug transport agents in vivo anti-cancer drug delivery

3.2. Collagen Based NPs in TE

Gold NPs were incorporated into collagen nanoparticle scaffolds which later on re-
acted with growth factors and molecules for cell adhesion. This helped in reduction of
inflammation and formation of granulation tissue with no problems of rejection, which
make them perfect for wound healing [32]. Volkova et al. conducted experiments which
displayed that gold NPs along with cryopreserved human fibroblasts administered top-
Appl. Sci. 2021, 11, 11369 12 of 17

ically to burns and wounds increased the rate of healing and enhanced collagen deposi-
tion [32,78]. Vedhanayagam et al. fabricated scaffolds based on silver, pectin and collagen
NPs which supplied greater antibacterial activity and improved viability of the cell toward
keratinocytes [79]. Patrascu et al. prepared scaffolds of silver and collagen NPs for burn
and wound healing, and skin repair. Hydroxyapatite silver collagen nanoparticle com-
posite scaffolds acted as prospective bone graft materials [80,81]. Nidhin et al. developed
a construct of collagen NPs for TE applications and imaging by crosslinking α-Fe2 O3
NPs capped with starch to collagen. This construct had enhanced mechanical properties,
had better crosslinking, was able to provide better viability to the cell, had greater super
paramagnetic behaviour and could be used in imaging and as bio implants [82,83].
Moon du et al. prepared tissue constructs based on skeletal muscles with acellular
tissue scaffolds based on collagen NPs which enhanced contractile force generation [80,84].
Wang et al. studied the production of collagen nanoparticle bone and development for
preservation of alveolar ridge. For protection of the residual ridge after extraction of the
tooth, artificial collagen nanoparticle bone was introduced into the patient. Scans taken
showed that the residual ridge had combined with the collagen nanoparticle bone which
also had a greater alveolar bone mineral density [35,85]. Shen et al., after trials on rabbits,
displayed enhanced rate of solid fusion and bone mineral density when bone based on
collagen NPs along with stem cells derived from adipose and allogene was utilized. They
could also be used to repair defects in the human ulna [86]. Quinlan et al. prepared porous
scaffolds using collagen glycosaminoglycan (CG) and bioactive glass to promote TE in the
bone [87,88]. Collagen and hydroxyapatite could be manufactured to form a composite
of collagen-hydroxyapatite by lyophilization and dehydrothermal treatment [89,90]. For
implants in vascular medicine, collagen NPs are used as a scaffold in tissue engineered
vascular grafts with the addition of different compounds. Collagen NPs behave as a
template for growth into the vascular graft and cell recognition [91].
Injections based on collagen NPs can be provided to locally deliver therapeutic compo-
nents or drugs to hinder neurodegeneration and they contribute structural foundation [70].
Zhang et al. showed the usage of collagen nanoparticle or nano-sized β-tricalcium phos-
phate conduits along with filaments of collagen and nerve growth agents for regeneration
of facial nerves [90]. Amiri et al. conducted an experiment to study the effects of cell
attachment of collagen NPs on crosslinked electrospun nanofibers. Collagen NPs enhanced
various properties such as viability of the cell, adhesion and more spreading on the scaffold.
Regarding crosslinking with glutaraldehyde, collagen NPs mimicked the extracellular ma-
trix better than collagen nanofibers [91]. Renal cartilage sponge collagen NPs were utilized
as an osmotic accelerator for hormone replacement therapy for transdermal delivery of
17β-estradiol-hemihydrate. Conclusions displayed that the hydrogels in which estradiol
collagen NPs were present prolonged the release time of estradiol and improved the ab-
sorption of estradiol to a great extent. Hence, sponge collagen NPs can act as prospective
carriers for transdermal drug delivery [92,93]. These investigations on collagen in TE are
summarized in Table 3.
Table 3. Investigations on Collagen NPs in TE.

Agent (Crosslinking/
Effect of Collagen NPs Application Reference
Stabilising/Optimising)
Reduction of inflammation and formation of
Gold NPs Wound healing [31]
granulation tissue with no problems of rejection
Gold NPs and
Increased rate of healing and enhanced collagen
cryopreserved human Burn and wound healing [80]
deposition
fibroblasts
Greater antibacterial activity and improved
Silver and pectin Dermal TE [81]
viability of the cell toward keratinocytes.
Burn and wound healing, skin
Hydroxyapatite and silver [82]
repair, bone graft materials
Appl. Sci. 2021, 11, 11369 13 of 17

Table 3. Cont.

Agent (Crosslinking/
Effect of Collagen NPs Application Reference
Stabilising/Optimising)
Enhanced mechanical properties, better
α-Fe2 O3 NPs capped with
crosslinking, better viability to the cell, greater Imaging, bio implants [83]
starch
super paramagnetic behaviour
Tissue constructs based on skeletal
Enhanced contractile force generation muscles with acellular tissue [84]
scaffolds
Protection of residual ridge after
Greater alveolar bone mineral density tooth extraction, artificial collagen [85]
nanoparticle bone.
Stem cells derived from Enhanced rate of solid fusion and bone mineral Repairing defects in the human
[86]
adipose and allogene density ulna
Collagen
glycosaminoglycan (CG) Bone TE [87]
and bioactive glass
For implants in vascular medicine,
Acted as template for growth into the vascular
scaffold in Tissue Engineered
graft and cell recognition
Vascular Grafts
Nano-sized β-tricalcium
phosphate and nerve Regeneration of facial nerves [92]
growth agents
Enhance cell viability, adhesion and more Mimicking extracellular matrix
Glutaraldehyde [93]
spreading on the scaffold (ECM)
Osmotic accelerator for hormone
replacement therapy for
Prolonged release time of estradiol and
transdermal delivery of
improved absorption of estradiol
17β-estradiol-hemihydrate, carriers
for transdermal drug delivery

4. Conclusions
Collagen is one of the most utilized biodegradable polymers as it provides ideal
polymeric aid for DDS, while behaving like a natural biomaterial with wound healing
and haemostatic properties [7]. Collagen-based biomaterials are extremely important
for regenerative medicine and TE. Due to its decreased immunogenicity and exceptional
biocompatibility, collagen is the selected protein for the fabrication of biomaterials [94].
Enhancement of mechanical strength, biodegradability and delivery characteristics support
the optimization of biomaterials based on collagen for biomedical applications [95]. The
experimental studies carried out by researchers, some of which have been reviewed in
this paper, demonstrated that in vivo degradability, absorption and drug delivery are
modulated by the physical or chemical crosslinking of collagen, to regulate the effect of
drug delivery [96]. Collagen NPs or nanospheres are thermally stable particles which
promptly attain their sterilization and act as an effective carrier for therapeutic compounds
and cytotoxic agents [97]. Due to their reduced size, high capacity of adsorption, water
dispersion ability to form clear colloidal solutions and high surface area, collagen NPs
display high potency to be utilised as formulations for regulated release of steroids or
antimicrobial agents [7]. Methods of nanoparticle preparation such as polyelectrolyte
complexation and desolvation are widely used, whereas nano spray drying is up and
coming. As mentioned in Table 1, all methods have their limitations and benefits and,
therefore, there is potential for carrying out vast research to overcome these disadvantages.
Drug release efficacy and drug loading of proteins are also adjusted based on nanoparticle
characteristics or drug type and concentration. Efficiency of drug transfer can be increased
Appl. Sci. 2021, 11, 11369 14 of 17

by controlling and maintaining features such as surface charge, shape and size. To produce
ideal collagen NPs, the correct process and materials should be selected according to
the relevant application [31]. In the coming years, smart collagen NPs will be able to
engage and guide stem cells to specific body sites and dictate the development of in vivo
tissues [98]. Collagen plays a crucial role in biomedicine and it is clear from the facts and
discussion in this review that collagen NPs are up and coming materials in drug delivery,
formation of tissues, proliferation and attachment of cells [34,99,100].

Author Contributions: Conceptualization, A.L. and A.A.; methodology, P.M.; software, A.A.; valida-
tion, A.L., P.M. and A.A.; formal analysis, A.A.; investigation, A.L.; resources, P.M.; data curation,
A.A.; writing—original draft preparation, P.M. and A.A.; writing—review and editing, A.L. and S.R.;
visualization, A.L.; supervision, A.L. and S.R.; project administration, H.L., S.R.; funding acquisition,
S.R., H.L. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Acknowledgments: The authors would like to thank Manipal Institute of Technology and Manipal
Academy of Higher Education for providing them with assistance and resources to help develop
their research interests.
Conflicts of Interest: The authors declare no conflict of interest.

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