1521-0081/67/3/541–561$25.00 http://dx.doi.org/10.1124/pr.113.008367PHARMACOLOGICAL REVIEWS Pharmacol Rev 67:541–561, July 2015Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics

ASSOCIATE EDITOR: MARKKU KOULU

Novel Delivery Systems for Improving theClinical Use of Peptides

Miia Kovalainen, Juha Mönkäre, Joakim Riikonen, Ullamari Pesonen, Maria Vlasova, Jarno Salonen,Vesa-Pekka Lehto, Kristiina Järvinen, and Karl-Heinz Herzig

Institute of Biomedicine and Biocenter of Oulu, Faculty of Medicine (M.K., K.-H.H.) and Medical Research Center Oulu and Oulu University Hospital(K.-H.H.), Oulu, Finland; Department of Applied Physics, Faculty of Science and Forestry (J.R.), Department of Applied Physics, Faculty of Science andForestry (V.-P.L.), and School of Pharmacy, Faculty of Health Sciences (M.V., K.J.), University of Eastern Finland, Kuopio, Finland; Department ofPharmacology, Drug Development and Therapeutics (U.P.), and Department of Physics and Astronomy, Faculty of Mathematics and Natural Sciences

(J.S.), University of Turku, Finland; and Leiden Academic Center for Drug Research, Leiden University, Leiden, The Netherlands (J.M.)

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542II. Peptide Drugs Approved for Clinical Use. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542

A. Background of Clinical Peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5421. Osteoporosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5442. Gastrointestinal System.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5443. Infection: Antimicrobial Peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5454. Cardiovascular System.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5455. Endocrine System.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5456. Cancer.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545

B. Therapeutic Targets of Peptide Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545C. Peptide Pharmacokinetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546

1. Absorption. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5462. Distribution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5473. Elimination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547

III. Peptide Delivery Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547A. Development Challenges of Peptide Delivery Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548B. Parenteral Peptide Delivery Systems In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549

1. Peptide Delivery Formulations for Intravenous Delivery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5492. Peptide Delivery Formulations for Subcutaneous Delivery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5493. Peptide Delivery Formulations for Intranasal, Pulmonary, and Transdermal Delivery. . 549

a. Intranasal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549b. Pulmonary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 552c. Transdermal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 552

IV. Porous Silicon as a Novel Material for Peptide Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553A. Fabrication and Properties of Porous Silicon. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553B. Porous Silicon as a Peptide Delivery System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554

V. Immunogenicity and Adverse Effects of Parenteral Peptide Delivery Systems . . . . . . . . . . . . . . . . . 556A. Immunogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556B. Adverse Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556

VI. Future Potential of Peptide Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558

The study was supported in part by the FinNano Project “PEPBI” (Enhanced therapeutic effects via intelligent peptide-loadednanoparticles) of the Academy of Finland (Grants 118002 and 277190); the Finnish Cultural Foundation; and the strategic funding ofUniversity of Eastern Finland (NAMBER [Novel Nanostructured Materials for Pharmaceutical, Biomedical and Environmental Applications]consortium).

Address correspondence to: Dr. Miia Kovalainen or Prof. Karl-Heinz Herzig, Institute of Biomedicine and Biocenter of Oulu, Faculty ofMedicine, University of Oulu, Aapistie 5; 90014 University of Oulu, Finland. E-mail: Miia.Kovalainen@oulu.fi or karl-heinz.herzig@oulu.fi

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Abstract——Peptides have long been recognized asa promising group of therapeutic substances to treatvarious diseases. Delivery systems for peptides have beenunder development since the discovery of insulin for thetreatment of diabetes. The challenge of using peptides asdrugs arises from their poor bioavailability resulting fromthe low permeability of biological membranes and theirinstability. Currently, subcutaneous injection is clinicallythe most common administration route for peptides. Thisroute is cost-effective and suitable for self-administration,and the development of appropriate dosing equipmenthas made performing the repeated injections relativelyeasy; however, only few clinical subcutaneous peptidedelivery systems provide sustained peptide release. Asa result, frequent injections are needed, which may causediscomfort and additional risks resulting from a poor

administration technique. Controlled peptide deliverysystems, able to provide required therapeutic plasmaconcentrations over an extended period, are needed toincrease peptide safety and patient compliancy. In thisreview, we summarize the current peptidergic drugs,future developments, and parenteral peptide deliverysystems. Special emphasis is given to porous silicon,a novel material in peptide delivery. Biodegradable andbiocompatible porous silicon possesses some uniqueproperties, such as the ability to carry exceptional highpeptide payloads and to modify peptide releaseextensively. We have successfully developed poroussilicon as a carrier material for improved parenteralpeptide delivery. Nanotechnology, with its differentdelivery systems, will enable better use of peptides inseveral therapeutic applications in the near future.

I. Introduction

Current typical drugs in clinical use have a small-molecular-weight compound (molecular mass,500 g/mol)that is absorbed into systemic circulation from thegastrointestinal tract after administration as an oralformulation. The number of approved biologic drugs(including vaccines and diagnostic proteins) is steadilyincreasing, however, from nine in 2010 to 18 in 2013. In2014, there were 11 approvals [Center of BiologicsEvaluation and Research, U.S. Food and Drug Admin-istration (FDA)].Peptides comprise amino acids connected by amide

bonds. The shortest natural peptide, thyrotropin-releasinghormone, is only three amino acids long. Oligopeptides orpeptides are usually shorter than 50 amino acids, butsometimes even 100-amino-acid-long chains are consid-ered peptides (Latham, 1999; Sato et al., 2006; McGregor,2008). In this review, peptides with a maximum of100 amino acids are considered.Physicochemical properties of macromolecules include

high molecular weight, varying aqueous solubility, andrapid degradation in the body, which inherently hindertheir efficient oral absorption. As a result, they areadministered frequently as parenteral injections, themost common example being the subcutaneous injectionof insulin. This review covers the current state-of-the-artof clinical peptides, with an outlook on peptide drugs inthe pipeline and parenteral peptide delivery systems.Special interest focuses on the novel, biodegradable, andbiocompatible drug carrier material, porous silicon, whichhas shown potential for clinical applications.Peptides can be used in the treatment of various

diseases, including endocrine dysfunctions, infectiousdiseases, cancer, central nervous system disorders, andgastroenterologic diseases (Stevenson, 2009; Malavolta

and Cabral, 2011). Already more than 100 peptide-baseddrugs have reached the market, and hundreds ofpeptidergic compounds are in clinical or preclinicalstudies (Lien and Lowman, 2003; Bellmann-Sickert andBeck-Sickinger, 2010; Vlieghe et al., 2010; Craik et al.,2013). The advantages and disadvantages of peptides asdrugs are summarized in Table 1. Compared with low-molecular-weight drugs, peptides can be more potent,more efficient, and more target specific (Vlieghe et al.,2010). Peptides are often better tolerated and may enableefficient replacement treatment; the adverse effects arerelated to their excessive pharmacodynamics Leaderet al., 2008; Bellmann-Sickert and Beck-Sickinger, 2010).With their gentler side effect profile and greaterselectivity and efficiency, peptides can easily be used indifferent pathologic states, including cancer. In theUnited States, peptides have reached the market fastercompared with other compounds because the clinical andapproval phases have been shorter (Reichert, 2003;Leader et al., 2008; Albericio and Kruger, 2012).

To enable the efficient use of peptides as drugs,various methods have been developed (Fig. 1). Thesizes of the different peptide delivery systems vary butcould extend to nanosized systems and peptide con-jugates. Nanotechnology can be used to influence drugcharacteristics, such as solubility, distribution, elimi-nation, or drug release. Some therapeutics exploitingnanotechnology are already on the market (Zhanget al., 2008; Malam et al., 2011).

II. Peptide Drugs Approved for Clinical Use

A. Background of Clinical Peptides

The FDA has approved 61 new biologic drugs since2000, which constitute nearly 20% of the totalapprovals (Mullard, 2014). Some biologic drugs have

ABBREVIATIONS: ADME, absorption, distribution, metabolism, and excretion; AUC, area under the curve; FDA, U.S. Food and DrugAdministration; GLP-1, glucagon-like peptide-1; PEG, polyethylene glycol; PLGA, poly(lactic-co-glycolic acid); PSi, porous silicon; PTH,parathyroid hormone; RBC, red blood cell.

542 Kovalainen et al.

been financially successful, and several reached theblockbuster category, including insulin glargine ($6.510million in 2012) and several antibody products (Craiket al., 2013). The most studied and well known exampleof a peptide in therapeutic use is the parenteralformulation of insulin. Several other peptide drugs arein clinical use and have a similar sequence as theendogenous hormones or neurotransmitters. The mostwell known are oxytocin for labor induction, somatotro-pin for growth control, and vasopressin to increase waterretention. One possibility to improve pharmacokinetic orpharmacodynamics properties is to modify the naturalpeptide sequence. Examples of this strategy are theglucagon-like peptide-1 (GLP-1) analog liraglutide andgrowth hormone analog octreotide for acromegaly(Stevenson, 2009). These peptides mimic the activityand structure of the endogenous ligand but have modifiedamino acid backbone or include amino acids to decreasetheir degradation. Many of the peptidergic drugs entering

the market at the moment contain modified amino acidsor cyclic structures for improved ADME (absorption,distribution, metabolism, and excretion) properties.

Recombinant protein expression, protein purifica-tion, and chemical peptide synthesis techniques havesignificantly boosted the development of peptide drugs.The manufacturing of full-length human peptides over-came economic and immunologic restraints by the ex-tracted animal peptides. Manufacturing of peptideagonists is more feasible and cheaper than manufactur-ing of antibodies or antagonists, seen in the 21st centuryin peptidergic drug launches (Table 2).

The major therapeutic area of peptides is metabolicdiseases (25%) (Albericio and Kruger, 2012). Insulin wasthe first peptide drug introduced for use in patients in1922 (Banting et al., 1922). The first product fortherapeutic use was bovine insulin, followed by protamineand zinc insulin (Grunberger, 2013), and the firstrecombinant human insulin produced reached the market

TABLE 1Pros and cons of using peptides as therapeutic compounds

Pros Cons

Various therapeutic targets UnstableGood selectivity Poor permeability through biologic membranesGood potency Very low oral bioavailabilityWell tolerated Variable solubilityLower toxicity High molecular sizeLow accumulation Often need for invasive administrationNo toxic metabolites Possible immunogenicityLower manufacturing costs compared with proteins

Fig. 1. Delivery systems for peptide release.

Peptide Delivery Systems 543

in 1980s (Keen et al., 1980; Clark et al., 1982). Insulinlispro was the first insulin analog created in 1996,followed by insulin aspart and glulisine in 2000 and2004, respectively (Grunberger, 2013). Long-acting in-sulin products glargine and detemir were then launchedsimultaneously. The first noninvasive insulin adminis-tration technique was introduced by the launch ofExubera (Pfizer and Nektar Therapeutics, San Francisco,CA), the inhalable insulin, in 2006, but it was available onthe market for less than a year because of low sales(Kling, 2008; Antosova et al., 2009). In addition, the FDAraised a serious concern related to the significant risk oflung cancer in Exubera users with a history of cigarettesmoking (http://www.fda.gov/Safety/MedWatch/Safe-tyInformation/Safety-RelatedDrugLabelingChanges/ucm122978.htm). In 2015, a new inhalable insulin,Afrezza, was launched in the United States by Sanofiand Mannkind Corporation. Other examples of success-ful peptidergic drugs are glucagon and GLP-1 agonists,like exenatide, liraglutide, pramlintide, and lixisenatide.Another peptide, related to energy metabolism, isa recombinant form of leptin, metreleptin, and wasrecently approved for clinical use for generalized lipodys-trophy and type 1 diabetes (Chou and Perry, 2013; Sinha,2014).

As mentioned earlier, peptide drugs are usuallydelivered via parenteral routes, but there are someexceptions. Cyclosporine, a cyclic decapeptide withpoor aqueous solubility belonging to class IV in thebiopharmaceutical classification, is one of these excep-tions, and formulations with improved bioavailabilityhave been developed using microemulsion technologies(Talegaonkar et al., 2008).

In addition to metabolic diseases, peptides are alsoused as therapeutics in disease in other organ systemsas follows.

1. Osteoporosis. Osteoporosis is an important areafor peptide therapeutics. Previously, salmon calcitoninwas available as an intranasal spray, but chronic use ofcalcitonin was associated with an increased risk ofcancer; hence, the formulation was withdrawn in 2012,leaving only injectable preparations for short-term use(http://www.emea.europa.eu/docs/en_GB/document_library/Press_release/2012/07/WC500130122.pdf). In addition,parathyroid hormone is used for treating osteoporoticpatients with a high risk of bone fractures. Besides thenative hormone, the recombinant human parathyroidhormone analog teriparatide can be used as subcutaneoustreatment daily (http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Summary_for_the_public/hu-man/000425/WC500027996.pdf).

2. Gastrointestinal System. Two peptide-based drugshave been recently approved to treat gastrointestinaldiseases. Linaclotide, a 14-amino-acid peptide agonist ofthe guanylate cyclase 2C has been licensed for treatingchronic constipation. The GLP-2 analog teduglutide islicensed for treating short-bowel syndrome and was the

TABLE

2Pep

tide

therap

eutics

appr

oved

in20

12–20

14in

theEur

opea

nUnion

orUnitedStatesor

both

Pep

tide

AA

Mod

eof

Action

Indication

Formulation

App

rova

lCom

men

t

Lucinactant

(Sinap

ultide

)21

Sur

factan

tRespiratory

distress

synd

rome

inpr

ematur

einfants

Inha

lation

2012

U.S.Orp

hanindication

Pep

tide

mim

icof

surfactant-

associated

proteinB

Peg

inesatide

Dim

eric

21+21

EOO

receptor

agon

ist

Ane

mia

inpa

tien

tswith

chronickidn

eydiseas

es.c.,i.v

.20

12U.S.(w

ithd

rawn20

13for

fatalhy

persen

sitivity

reaction

s)Orp

hanindication

PEGylated

EPO-m

imetic

peptide

Pas

ireotide

6Som

atostatinreceptor

agon

ist

Cus

hing

diseas

es.c.

2012

EU,U.S.Orp

han

indication

Cyclohex

apep

tide

analog

ofsomatostatin

Carfilzom

ib4

Chym

otryps

in-likepr

otea

seinhibitor

Multiplemye

loma

i.v.

2012

U.S.Orp

hanindication

Ana

logof

epox

omicin

Linaclotide

14Gua

nylate

cyclas

e-C

agon

ist

Con

stipation-pr

edom

inan

tirritablebo

wel

synd

rome,

chronicidiopa

thic

cons

tipa

tion

Oral

2012

EU,U.S.

3Cys-bridg

es

Ted

uglutide

33GLP-2

receptor

agon

ist

Sho

rtbo

wel

synd

rome

s.c.

2012

EU,U

.S.O

rpha

nindication

Glycine

mod

ified

Lixisen

atide

44GLP-1R

agon

ist

Typ

e2diab

etes

s.c.

2013

EU

Mod

ifiedex

endin-4

Afamelan

otide

13MC1R

agon

ist

EPO

protop

orph

yria

(pho

topr

otectant)

s.c.

implan

t20

14EU

Orp

hanindication

a-M

SH

analog

withmod

ified

AAs

Albiglutide

30GLP-1R

agon

ist

Typ

e2diab

etes

s.c.

2014

EU,U.S.

GLP-1

fusedto

human

albu

min

Dulaglutide

46GLP-1R

agon

ist

Typ

e2diab

etes

s.c.

2014

EU,U.S.

GLP-1

fusedto

human

Fc

frag

men

tMetreleptin

Lep

tinan

alog

Gen

eralized

lipo

dystroph

ys.c.

U.S.

AA,n

umbe

rof

aminoacids;

EPO,e

rythropo

ietin;G

LP-1R,glucago

n-likepe

ptide1;

MC1R

,melan

ocortin-1

receptor;M

SH,m

elan

ocyte-stim

ulating

hormon

e.

544 Kovalainen et al.

first long-term therapy for treating patients dependent onparenteral nutrition (Burness and McCormack, 2013).3. Infection: Antimicrobial Peptides. Antibiotic re-

sistance is a growing threat in health care. Theendogenously released antimicrobial peptides witheffects against bacteria, fungi, protozoa, and virusesare promising pathways to combat nosocomial infec-tions (Anglin et al., 2004; Fox, 2013; Gaspar et al.,2013). Daptomycin was introduced to European andUS markets in 2006 and 2003, respectively (http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Summary_for_the_public/human/000637/WC500036050.pdf). Gramidicin and bacitracin areantibacterial polypeptides that are used locally, forexample, locally in topical and eye infections. Caspo-fungin is available for treating invasive Candidainfection as an intravenous infusion.4. Cardiovascular System. Several peptide-based

drugs have been developed for cardiovascular diseases,including anticoagulants like bivalirudin and epifibatige.Bradykinin B2 receptor competitive antagonist icatibantis a decapeptide used as subcutaneous treatment ofhereditary angioedema. There are also several recombi-nant forms of human endogenous peptides on the marketas intravenous preparations, such as atrial natriureticpeptide carperitide and B-type natriuretic peptide nesiri-tide for different types of heart failure.5. Endocrine System. Somatostatin and analogs such

as depreotide, lanreotide, pentetreotide, and octeotrideare used for treating acromegaly and variceal bleeding, aswell as for diagnostic purposes. Previously, an erythro-poietin analog, peginesatide, was available for treatmentof chronic kidney disease patients with anemia, but it waswithdrawn in 2013 for safety issues. Vasopressin andmodified desmopressin, as well as terlipressin, are avail-able for treating diabetes insipidus and hepatorenalsyndromes.6. Cancer. Anticancer drugs are the second largest

group of peptide therapeutics (16%) in clinical use,most for prostate cancer. Among the newest approvedoncologic drugs are brentuximab vedotin for Hodgkinlymphoma and romidepsin for cutaneous T-cell lym-phoma (Albericio and Kruger, 2012). Leuprorelin,goserelin, triptorelin, and buserelin have been on themarket longer for treating prostate cancer as implant-able delivery systems. Another recently approvedpeptide for prostate cancer treatment is degarelix.Bortezomib has been available since 2004 and 2008 inEurope and the United States, respectively, andcarfilzomib was approved in 2012 by the FDA fortreating patients with multiple myeloma (http://www.fda.gov/AboutFDA/CentersOffices/OfficeofMedicalPro-ductsandTobacco/CDER/ucm094633.htm; http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Summary_for_the_public/human/000539/WC500048136.pdf; http://www.accessdata.fda.gov/drugsatfda_docs/la-bel/2012/202714lbl.pdf).

B. Therapeutic Targets of Peptide Drugs

Already more than 400 peptide-based drugs (definedhere as ,100 amino acids) were reported to be in thepipelines at the beginning of the year 2012 (Kaspar andReichert, 2013). Pipeline analyses show that the toptherapeutic areas are metabolic diseases, oncology, andinfectious diseases (Kaspar and Reichert, 2013). Approx-imately 10% of the peptidergic pipeline is in the area ofantimicrobial peptides. The most frequent single-proteintarget for peptide therapeutics in clinical studies isGLP-1R, which has five drugs launched, two in prereg-istration, 19 in clinical studies, and an additional 19 inpreclinical studies (Citeline, Jan-14; https://citeline.com/products/pharmaprojects/).

Unlike with traditional drug compounds, the pharma-cologic effects of peptides often regulate complex endog-enous physiologic pathways. The responses may beindirect and involve tolerance, rebound phenomena,and negative feedback controls, which have an increasedinterest in using physiologically-based pharmacokineticmodeling for peptide therapeutics (Diao and Meibohm,2013). Many of the current drugs that target membraneproteins, such as G protein–coupled receptors, sufferfrom poor specificity causing inadequate signaling effects.The natural and synthetic therapeutic peptides are oftenpotent receptor agonists, requiring lower concentrationsfor receptor activation (Hruby, 2002; Lien and Lowman,2003). Peptides can also be used as replacement therapywhen the endogenous peptide system is deficient (Leaderet al., 2008; Bellmann-Sickert and Beck-Sickinger, 2010).

GLP-1 analogs exenatide, liraglutide, and the newest,lixisenatide, are examples of peptide drugs that enhancethe effects of existing functional pathways to achieve thedesired therapeutic effects. Those peptide drugs produceincretin-like effects, including the induction of glucose-dependent insulin secretion and the inhibition ofglucagon release, slowing of gastric emptying, andinduction of a sensation of fullness, all of which resultin improved glucose balance and weight loss in type 2diabetic patients (Elkinson and Keating, 2013). They actvia G protein–coupled GLP-1 receptors in the pancreaticislet cells, causing protein kinase B activation and insulinsecretion (Ahren, 2009). A recently approved peptidedrug, teduglutide, is used in short-bowel syndrome byactivating the GLP-2 receptor. It increases the release ofvarious mediators, for example, nitric oxide, keratinocytegrowth factor, and insulin-like growth factor-1 (Burnessand McCormack, 2013). Peptides used for in vitrofertilization, such as human follicle-stimulating hormoneor teriparatide for the treatment of osteoporosis, alsostimulate the action of existing pathways. Parathyroidhormone (PTH) is the endogenous regulator of calciumand phosphate. Teriparatide is a 34-acid fragment of thePTH, consisting of 84 amino acids, that simulates thephysiologic action of PTH, resulting in increased intestinalabsorption and tubular reabsorption of calcium and

Peptide Delivery Systems 545

improvement in bone formation (http://www.accessdata.fda.gov/drugsatfda_docs/label/2008/021318s015lbl.pdf).The mechanisms of actions of peptides are not always

receptor mediated, for example, lucinactant, which is usedas a surfactant in prematurely born infants to preventrespiratory distress syndrome (Table 2). Anticancerpeptides have oncolytic, antiproliferative, and proapop-totic functions in tumor cells, which may be membrano-lytic or nonmembranolytic mechanisms (Gaspar et al.,2013). Many of the anticancer peptides in clinical use bindto the 20S proteasome, inhibiting its activity.

C. Peptide Pharmacokinetics

Pharmacokinetic properties of peptides are settinga major challenge for the clinical use of many peptides.Recently, some excellent reviews focusing solely onpharmacokinetics of peptides (Lin, 2009; Diao andMeibohm, 2013) have been published; hence, only thegeneral aspects of peptide pharmacokinetics arereviewed here.1. Absorption. Transport of peptides across biologic

membranes is severely limited by their large molecularsize (.500 Da) and hydrophilicity. The absorption stepis avoided only in intravenous administration, but inother administration routes, peptides need to crossseveral biologic barriers, such as epithelia or themucosal layer. The structural and metabolic differencesin biologic barriers result in substantial differences inpeptide absorption.The subcutaneous administration route is the most

commonly used way of delivering peptides (Lin, 2009)and is more convenient for administration than theintravenous route, primarily for safety reasons; however,local blood flow, injection depth and volume, peptidedegradation in the subcutaneous tissue, and the molec-ular weight of the peptide all can affect the absorption ofthe peptide (Tang et al., 2004; Richter et al., 2012).Although subcutaneous administration is the mostcommon route, many parameters affecting subcutaneousabsorption of peptides or proteins are not completelyunderstood.In sheep, small peptides (,1 kDa) are absorbed into

the systemic circulation, whereas a significant portion(20%–40%) of larger peptides and small proteins (5–12 kDa)are absorbed via the lymphatic system (Supersaxoet al., 1990; Charman et al., 2000; Lin, 2009). Lymphaticvessels have a higher permeability than blood capil-laries, thus allowing better penetration for largermolecules. In rats, Kagan et al. (2007) found practicallyno insulin absorption (,0.1%) into the lymphaticsystem after subcutaneous administration. Therefore,more information is needed to understand the differ-ences between animal models and experimental set-tings (Lin, 2009; Richter et al., 2012).In other parenteral but noninvasive administration

routes, peptides need to cross the physical barrierspresented by epithelia cells, dynamic and steric barriers

by mucus, or metabolizing enzymes on the surface ofepithelia. Three routes are available for peptideabsorption through epithelia, similar to other drugs orxenobiotics, namely, 1) through the cells (transcellular),2) through intercellular junctions (paracellular), or 3) byactive transport or receptor-mediated mechanisms(Burton et al., 1996; Boguslavsky et al., 2003). It istypically proposed that most peptides are absorbed viathe paracellular route (Patton, 1996; Veuillez et al.,2001; Lin, 2009; Ozsoy et al., 2009).

The most important factors limiting the permeabilityof peptides through the epithelia are an increasednumber of hydrogen bonds, hydrophilicity, and molec-ular size greater than 700 Da (Burton et al., 1996;Ramaswami et al., 1996; Lin, 2009; Ozsoy et al., 2009;Diao and Meibohm, 2013). The transcellular transportacross the lipophilic cell membranes is limited by thepeptide’s polarity, and paracellular permeation is re-stricted by their large size (He et al., 1998; Lin, 2009).Paracellular permeation has been estimated to belimited to the molecules with molecular mass #3.5 kDain intestinal epithelia (Madara and Dharmsathaphorn,1985; Rubas et al., 1996). Absorption enhancers areusually needed for efficient peptide absorption when thesize of the peptide exceeds 0.5–1 kDa (Borchardt et al.,1997; Veuillez et al., 2001; Ozsoy et al., 2009); however,the intranasal route can allow absorption of largerpeptides (.2 kDa) without absorption enhancers (Tanget al., 2004), and the pulmonary route via endothelialjunctions can allow absorption molecules in size of4–6 nm (Sayani and Chien, 1996). Besides the size, theshape of the peptide has an effect on its ability topermeate through barriers. The permeability of rigidcyclic peptides through biologic membranes is lowerthan that of more flexible linear peptides (Boguslavskyet al., 2003; Kwon and Kodadek, 2007).

Epithelia are significant absorption barriers for peptideabsorption, but there are significant differences betweenadministration routes. Barrier function is the weakest inlung alveoli, where epithelia are only 0.2 mm thick. Thelarge surface area (100 m2), efficient vascularization ofthe lungs, and bypassing of the intestinal epithelium andliver metabolism enable good pulmonary bioavailability ofpeptides (Patton, 1996). In contrast, the top layer of theskin, the stratum corneum (thickness, 10–20 mm) limitsthe transdermal absorption of polar and large peptides(Benson and Namjoshi, 2008; Prausnitz and Langer,2008). Peptide permeability can also vary within admin-istration routes, such as in the oral cavity, wherepermeability is better in sublingual and buccal sites,which are nonkeratinized, compared with the keratinizedsites of the oral cavity (Veuillez et al., 2001).

Another factor limiting peptide absorption is themetabolic barrier caused by degrading enzymes on orinside epithelia. Enzymatic degradation also occurs insubcutaneous tissue, but the mechanisms are not com-pletely understood (Richter et al., 2012). The advantage of

546 Kovalainen et al.

nasal, pulmonary, and transdermal administration routesis that the hepatic first-pass metabolism can be avoided,which can significantly increase the peptide bioavailabil-ity (Moeller and Jorgensen, 2008). In the case ofpulmonary administration, the efficacy of the enzymaticbarrier is defined by the peptide size: peptidases degraderapidly smaller peptides (,3 kDa), but the pulmonaryantiproteases can protect larger peptides (e.g., calcitonin)from rapid degradation (Patton et al., 2004). In the nasalcavity, several endopeptidase and exopeptidase enzymeson the mucosal membrane affect peptide absorption(Veuillez et al., 2001; Costantino et al., 2007). Concerningthe dermal route, the physical barrier of the stratumcorneum is supported by the enzymatic degradation ofpeptides in epidermis and dermis (Shah and Borchardt,1991; Ogiso et al., 2000; Bachhav and Kalia, 2009). Innasal cavities and lung bronchi, the mucous layersrepresent a physical barrier with electrostatic interactionsbetween negatively charged mucus and positively chargedpeptides (Veuillez et al., 2001), whereas unabsorbedpeptides can be removed from the absorption site bymucociliary clearance.2. Distribution. Peptide distribution is determined

by molecular weight, charge, protein binding, anddependence of active transport. The distributionvolume is relatively small for peptides, approximately3–8 liters for the central compartment and 14–20 litersfor the steady-state phase (Tang et al., 2004; Diao andMeibohm, 2013). Small molecules and peptides (,500 Da)are distributed mainly by diffusion, which can alsooccur for larger proteins (.5–10 kDa), only moreslowly, through the nanopores of capillary walls(Grotte, 1956; Bill, 1977; Lin, 2009). Larger peptidescan be cotransported with water (Lin, 2009). Theinfluence of molecular weight on the distribution ratewas examined by studying extravasation time ofpolyamidomine dendrimers. The extravasation timewas exponentially increased as the molecular massincreased from 0.5 to 14.2 kDa (El-Sayed et al., 2001);however, few mechanistic studies have been reportedon this topic (Vugmeyster et al., 2012). Furthermore,binding of peptides to plasma proteins affects peptidedistribution: 65% of octreotide is bound to lipoproteinsand more than 98% liraglutide is bound to plasmaproteins (Diao and Meibohm, 2013).3. Elimination. Peptides are metabolized within

minutes (Diao and Meibohm, 2013), which explains theirshort elimination half-lives and therapeutic effects. Theproteolytic enzymes are mainly responsible for peptidemetabolism, and they can be found comprehensivelyfrom the whole system, but the most important organsare the liver, kidneys, and blood (Tang et al., 2004; Werleand Bernkop-Schnurch, 2006; Lin, 2009). Solubleenzymes, which are found in blood, or membrane-boundenzymes are frequently responsible for peptide metabo-lism (Werle and Bernkop-Schnürch, 2006). In contrast,cytoplasmic enzymes have often a minor role in the

peptide metabolism. The proteolytic enzymes can beclassified into exopeptidases and endopeptidases, whichhave different mechanism of action. Exopeptidases cleavea few amino acids from the N or C termini of the peptide,whereas endopeptidases degrade peptide bonds from themiddle (Werle and Bernkop-Schnurch, 2006; Lin, 2009).

Receptor-mediated peptide uptake is a specific featureof peptide elimination because the receptor saturationcan occur even at therapeutic concentrations, which canlead to nonlinear pharmacokinetics (Tang et al., 2004;Diao and Meibohm, 2013). Hepatic metabolism issignificantly less important for most peptides than forsmall-molecule drugs; however, some small peptides,such as cyclosporine and bortezomib, are metabolizedalmost completely in the liver (Diao and Meibohm, 2013).

Peptides are freely filtered by the kidney glomeruli,but this has minor significance in the overall elimina-tion of several peptides compared with metabolicdegradation (Tang et al., 2004; Werle and Bernkop-Schnurch, 2006). Only when the enzymatic degradationpathway is blocked is renal clearance a significantelimination pathway for peptides (Lin, 2009). Thus, inthe kidneys, small linear peptides (e.g., angiotensin Iand II or peptide YY 3-36 [PYY3-36]) are hydrolyzed onthe luminal membrane by brush-border enzymes(Carone and Peterson, 1980; Addison et al., 2011). Incontrast, peptides with higher molecular weight (e.g.,insulin) are subject to lysosomal degradation after theyhave been taken up by endocytosis (Carone et al., 1982).If peptides are resistant to proteolysis, renal filtrationmight have a significant role in their elimination. Thisis the case for exenatide, a GLP-1 mimetic, resistant fordipeptidyl peptidase IV, which increases eliminationhalf-life from 2 minutes of GLP-1 to 2.5 hours.

III. Peptide Delivery Systems

When using peptides as medication, it is usuallynecessary to adjust their pharmacokinetic character, forexample, to enhance their absorption or to prolong theirtime of action. The objective can be achieved by 1)stabilizing the peptide structure, 2) inhibiting thedegradation by suitable compounds, 3) improving theabsorption with help of aiding substances, or 4) gener-ating controlled peptide delivery systems protecting thepeptide (Frokjaer and Otzen, 2005).

Oral delivery is certainly the most desired butchallenging route and has been recently reviewed byRenukuntla et al. (2013); thus, it will not be discussedfurther here. Oral formulations of peptidergic drugstend to have limited bioavailability. If oral formulationsare not feasible, the alternative administration techni-ques should be as convenient as possible to ensure highpatient compliance. Since poor physical chemical andADME properties are immanent to peptides, effectivedelivery with controlled release and less invasivetechniques are needed.

Peptide Delivery Systems 547

The use and fabrication of traditional peptide deliverysystems have been recently reviewed elsewhere (Oaket al., 2012; Jain et al., 2013; Du and Stenzel, 2014;Mitragotri et al., 2014); therefore, we will give a moregeneral introduction to most commonly used (nano)materials in peptide delivery. There are certain chal-lenges or disadvantages related to the fabrication andstability of the peptides and delivery systems; these arediscussed later (Frokjaer and Otzen, 2005; Jain et al.,2013; Mitragotri et al., 2014). Examples of traditionalpeptide delivery systems are summarized in Table 3.Various polymer-based delivery systems have been

applied for peptides using both synthetic and naturalmaterials, including gelatin, hyaluronic acid, cellulose,chitosan, poly(lactic-coglycolic) acid (PLGA), polycapro-lactones, polyanhydrides, and cyclodextrins (Jain et al.,2013; Du and Stenzel, 2014; Mitragotri et al., 2014; Patelet al., 2014). The fabrication of polymer-based peptidedelivery systems can be performed by several techni-ques, such as phase separation, solvent evaporation, orspraying. The drug is equally distributed in the polymermatrix (Fig. 1). Drug release mechanisms vary accordingthe polymer type and structure and can be controlled bydifferent external stimuli (Oak et al., 2012).Microemulsions are surfactant-based drug carriers

that are considered “water-in-oil” or “oil-in-water” stableemulsions in which the particle size is usually fewnanometers. They have a hydrophilic part outside toachieve solubility and a hydrophobic core that allowsdissolution of hydrophobic compounds (Du and Stenzel,2014). Solid lipid nanoparticles consist of a lipid core forpeptide loading and a hydrophilic surfactant layerstabilizing the particle in an aqueous environment.The fabrication of solid lipid nanoparticles is notconsidered as stressful for the loaded compound incomparison with the most often used polymeric pro-duction techniques (Almeida and Souto, 2007); however,the technology may set limitations for hydrophilicpeptides (Du and Stenzel, 2014). In contrast to pre-viously described lipid-based delivery systems, whichconsist of hydrophobic core and hydrophilic shell,liposomes have an opposite structure. Liposomes arevesicles, which have a bilayer phospholipid membraneallowing entrapping hydrophilic peptides inside theprotective shell. As in the other delivery systems,liposomes protect the peptide from degradation, prolong

circulation time, and increase bioavailability (Patelet al., 2014). Liposomes have been demonstrated toserve as a carrier for various peptides, such as insulin,vasoactive intestinal polypeptide, and calcitonin (Duand Stenzel, 2014); however, some major issues relatedto their stability need to be resolved (Patel et al., 2014).

In conclusion, different delivery systems and materi-als have been investigated for peptides; however, eachcarrier has its own advantages and disadvantages, anda suitable carrier is often determined by the physico-chemical characters of the peptide, the desired route ofadministration, and clinical aspects of therapy.

A. Development Challenges of PeptideDelivery Formulations

Developing peptide delivery systems is often lessstraightforward compared with the formulation ofsmall-molecule compounds, and bioactivity is oftenjeopardized during the formulation process (Shire, 2009;Ye et al., 2010). The overall stability and the peptidestructure may be affected by various factors during theformulation process, e.g., heat, pH, strong solvents,contaminations, shaking, or storing (Wang, 2005; Yeet al., 2010; Jiskoot et al., 2012). As an example, lysozymelost almost completely its bioactivity in biodegradablemicrospheres as a result of the fabrication conditions(Ghaderi and Carlfors, 1997). Variable conditions in thepreparation of GLP-1 solutions significantly influencedthe onset of response after subcutaneous administrationabsorption rate and bioavailability by formed aggregates(Clodfelter et al., 1998). Furthermore, instability in theaqueous environment can be encountered as glucagonformed cytotoxic fibrillates, when stored for long periodsin concentrated solutions, and at high temperature (.37°C)(Onoue et al., 2004).

Challenges are also related to the use of different kindof particles to improve the peptide delivery. The in vitrorelease of calcitonin from poly(ethylene glycol)-terephthal-ate and poly(butylene terephthalate) delivery systemswas incomplete as a result of peptide aggregation causedby sodium that was used in the in vitro release test (vanDijkhuizen-Radersma et al., 2002). Three chitosan-basedoral delivery systems could demonstrate prolonged re-lease over 4 hours in vitro, but when tested in rats, onlyone was able to decrease significantly plasma calciumlevels in a sustained manner (12 hours) (Guggi et al.,

TABLE 3Examples of different peptide delivery systems

Drug Delivery System (Route) Peptide Outcome Reference

Unilamellar liposomes (inhalation) Vasoactive intestinal peptide Sustained release and extendedpharmacologic effect ex vivo

Hajos et al., 2008

Poly(lactide-co-glycolide)microspheres (i.m. )

Vapreotide Erosion-controlled release up to4 wk in vivo

Blanco-Prieto et al., 2000

Water in oil microemulsion(intranasal)

Insulin Relative bioavailability 21.5% to s.c.administration

Sintov et al., 2010

Solid lipid nanoparticleflocculates (pulmonary)

Insulin Relative bioavailability 35.6% to s.c.administration

Yang et al., 2012a

548 Kovalainen et al.

2003). PLGA is very commonly used polymer withpeptides, but its drawback is the potential of peptidedegradation reactions in the delivery system (Houchinand Topp, 2008). It is important to note that many of theearlier developed carrier materials often have a restrictedcapacity to carry cargo as the commonly investigatedmicroparticles have only approximately 7% peptide/protein content (Ye et al., 2010). In addition, the deliverysystems are often accompanied with significant burstrelease of the peptide (Ye et al., 2010).

B. Parenteral Peptide Delivery Systems In Vivo

1. Peptide Delivery Formulations for IntravenousDelivery. Intravenous injection is a commonly usedadministration route for peptide therapeutics (Fig. 2;Table 4). The advantages include bypassing of the first-pass metabolism and 100% bioavailability as there isno drug loss at the injection site as well as fast onset ofaction; however, it is invasive and requires health careprofessionals for its administration.To use the therapeutic effects of peptides, the aim is

to prolong their circulation time after intravenousadministration. In general, after intravenous injection,peptide drugs are widely distributed and rapidlyeliminated. Attempts have been made to address thechallenge of their short plasma half-life by use ofdifferent drug delivery systems. As a common ap-proach, the peptide is conjugated with a carrier, whichreleases the drug by different mechanisms and thusprotects the peptide from rapid degradation. Theeffects of GLP-1 and its half-life were improved bya delivery system in which protease activity releasesalbumin conjugated GLP-1 (Li et al., 2010). Anotherexample is polyethylene glycol (PEG) conjugation ofsalmon calcitonin, which resulted in prolonged half-lifebut affected its potency negatively (Ryan et al., 2009),suggesting that the improved circulation time may notalways improve the therapeutic effects.2. Peptide Delivery Formulations for Subcutaneous

Delivery. Table 5 presents a wide variety of differenttechnologies that have been investigated for subcutane-ous peptide delivery. The bioavailability of peptide drugsis variable after subcutaneous injection (Table 5), andneither the route nor how peptides are absorbed from thesubcutaneous tissue to bloodstream are entirely un-derstood and may be species dependent (Vugmeysteret al., 2012). As an illustration, two compounds showedlow bioavailability after subcutaneous administration:less than 20% for PYY3-36 and 40% for PEGylated form oferythropoietin in rats, whereas the bioavailability ofbuserelin has been reported to be 70% after subcutane-ous delivery to humans (Mönkäre et al., 2012; Vugmeysteret al., 2012; Wang et al., 2012). The absorption of somebiomolecules from subcutaneous injection site is veryefficient; as an example, the marketed insulin-likegrowth factor-1 product mecasermin (Increlex) achievesalmost 100% absolute bioavailability (Vugmeyster et al.,

2012). Flip-flop kinetics, where the drug is released fromits carrier, regulates the plasma concentration, therebyprolonging the presence of peptides in circulation (Diaoand Meibohm, 2013).

3. Peptide Delivery Formulations for Intranasal,Pulmonary, and Transdermal Delivery. Pulmonary,intranasal, and transdermal are other promising routesfor noninvasive drug delivery. Each of these routes hastheir distinct advantages and disadvantages based ontheir anatomic and physiologic features (Table 6). Recentexamples of different peptide delivery systems developedfor these routes and studied in vivo are summarized inTable 7.

a. Intranasal. The intranasal route has a highlyvascularized subepithelia layer and lower enzymaticactivity compared with the oral route, making it a moredesired administration route; however, the systemicbioavailability is often limited to 1%, and absorptionenhancers can be used to improve the efficiency of in-tranasal peptide delivery (Illum, 2012). Intranasally-administered peptide formulations that are or have beenon the market (calcitonin, buserelin, desmopressin,nafarelin, and oxytocin) do not contain any absorptionenhancers, most probably owing to the poor tolerability ofthe enhancers in the nasal cavity (Ozsoy et al., 2009;

Fig. 2. Delivery routes for peptides. LHRH, luteinizing hormone–releasing hormone; VEGF, vascular endothelial growth factor.

Peptide Delivery Systems 549

Illum, 2012). In addition, the intranasal route suffers fromlarge interindividual variability in absorption resultingfrom frequent pathologic nasal conditions (e.g., hay fever)(Lochhead and Thorne, 2012).Nasal absorption of peptides can be improved by

different strategies: 1) the promotion of the absorptionprocess by interacting with membrane, 2) prolonging theresidence time of the peptide formulation in the nasalcavity, or 3) inhibiting enzyme activity. Peptide penetra-tion through the nasal mucosa is the key factor for thenasal absorption of peptides, and it can be improved byusing mucoadhesion strategies. The residence time of theformulation on mucosa is important because the half-lifeof the clearance for nonadhesive formulations is only15–20 minutes. Many penetration enhancers have beenstudied clinically for peptide delivery, for example, cyclo-penta delactone (insulin) (Leary et al., 2006), hydroxylfatty acid esters of polyethylene glycol (CriticalSorb,insulin, teriparatide) (Lewis et al., 2009), alkylsaccharides

(cyclic PTH1-31) (Illum, 2012), and chitosan (Chisys,goserelin) (Illum, 2012). However, the development ofmany of these enhancers has been terminated or datahave not been published (Illum, 2012). CPEX Pharma-ceuticals (Exeter, NH) developed Nasulin spray forintranasal insulin administration using cyclopenta delac-tone to enhance its absorption (Leary et al., 2006). Therelative bioavailability to subcutaneous insulin in type 1diabetes patients was 17%–20% in the first 2 hours (Learyet al., 2005, 2006), but further development was halted in2010.

Because penetration enhancers can be toxic, micro-emulsions have been studied to improve intranasalabsorption of insulin because they have been suggestedto be better tolerated (Sintov et al., 2010). This approachwas successfully applied for delivery of insulin inrabbits. The relative bioavailability of insulin was21.5% using microemulsion resulting from enhancedintramucosal transport (Sintov et al., 2010). Another

TABLE 4Drug delivery systems for intravenous administration

Technique Peptide Drug Outcome Reference

Peptide drug is linked by aprotease-sensitive compoundto albuminbinding peptide

Glucagon-like peptide 1(GLP-17-37, 3.4 kDa)

Sustained the pharmacologic effect andprolonged half-life of elimination

Li et al., 2010

Peptide is conjugated using apoly(PEG) methyl ethermethacrylate with acomb shape

Salmon calcitonin(3.5 kDa)

Significantly prolonged half-life of elimination(even 15�) but a decrease in potency

Ryan et al., 2009

Adsorption of peptide intoporous silicon nanocarriers

Human peptide YY3-36 Successful delivery of an active peptidebut no improvement in circulation timeor pharmacokinetic parameters comparedwith peptide solution

Kovalainen et al., 2013

Immobilization of cysteineincluding peptide thiolatedcarboxymethyl dextran-cysteine conjugate

DALCE Five-fold improvement in elimination half-lifeand 6.7-fold decreased plasma clearance rate

Shahnaz et al., 2012b

DALCE, [D-Ala2, Leu5, Cys6]-enkephalin.

TABLE 5Different in vivo investigated peptide delivery systems for subcutaneous delivery

Technique Peptide drug Outcome References

mPEG-PLGA-mPEG-basedthermosensitive in situforming gel

Salmon calcitonin (mol. mass3.5 kDa)

Controlled delivery andpharmacologic activity for20–40 d

Tang and Singh, 2010

Noncovalent Zn-peptide adduct(spray dried)

BMS-686117 (mol. mass 1.5 kDa) Terminal half-life prolonged by6 h and Cmax values reduced6- to 8-fold compared withsolution

Qian et al., 2009

Solution Exenatide .100% (due to underestimationof i.v. AUC)

http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_Scientific_-_Discussion/human/000698/WC500051842.pdf

Thermoresponsive polylactic acid-polyethylene glycol-polylacticacid triblock copolymer

Insulin Controlled release of bioactivemolecule up to 3 mo

Al-Tahami et al., 2011

Aminoacid modification Insulin lispro 55–77% absolute bioavailability Vugmeyster et al., 2012Solution Liraglutide 55% absolute bioavailability Stevenson, 2009; Perry, 2011PLGA microspheres LXT-101 (LHRH antagonist) Sustained-delivery system,

efficacyDu et al., 2006

Porous silicon microparticles andnanoparticles

PYY3-36 Controlled-delivery system,increased bioavailability

Kovalainen et al., 2012, 2013

DALCE, [D-Ala2, Leu5, Cys6]-enkephalin; LHRH, luteinizing hormone–releasing hormone.

550 Kovalainen et al.

recent alternative to improve nasal absorption are un-saturated glycoceramides, which use endogenous sphin-golipid trafficking (te Welscher et al., 2014). Absorption ofGLP-1 linked to glycosphingolipid GM1 was enhanced,demonstrating therapeutic potential; yet there are chal-lenges in the efficient cleavage of the linkage between thepeptide and glycosphingolipid after absorption.Thiolated polymers have been used as potential

excipients for the nasal delivery since they are safe,improve residence time on the nasal mucosa, and act aspenetration enhancers and enzyme inhibitors (Vetter andBernkop-Schnurch, 2010; Vetter et al., 2010; Palmbergeret al., 2011). In rats, leuprolide-loaded thiolated chitosan

nanoparticles increased the area under the curve (AUC),Cmax, and elimination half-life after nasal application by6.9-, 3.8-, and 4-fold, respectively, compared with thesolution administration (Shahnaz et al., 2012a). Relativebioavailability was reported to be 19.6%. In the case ofnonthiolated chitosan nanoparticles, such an improveddelivery in comparison with the leuprolide solution wasnot seen. Earlier, thiolated chitosan microparticles havebeen also shown to improve intranasal delivery of insulin(Krauland et al., 2006).

Intranasal administration can offer enhanced cen-tral nervous system absorption via the olfactory region.This has been shown for insulin (Renner et al., 2012),

TABLE 6Key characteristics of different nonparenteral peptide administration routes

NonparenteralAdministration Routes Advantages Disadvantages

Intravenous Efficient systemic delivery Self-administration is not possibleSubcutaneous Relatively efficient systemic delivery. Clinical accepted Mechanisms affecting absorption are not well known

Injections are neededPulmonary First-pass metabolism avoided Failure of inhaled insulin

High surface area and vascularization, rapid absorption Ensuring efficient inhalationIntranasal First-pass metabolism avoided Variability of absorption (from, e.g., hay fever or

common cold)Intranasal peptide drugs on market.Possibility for central nervous system administration

via olfactory regionTransdermal Easy to access for patient Stratum corneum creates a strong absorption barrier

Suitable for sustained release

TABLE 7Examples of in vivo studies of peptide delivery systems for intranasal, pulmonary, and transdermal administration

Adapted largely from Mönkäre, 2012.

Technology Peptide (mol. mass) Result Reference

IntranasalWithout microemulsion to enhance absorption

without using irritating excipientsInsulin (5.8 kDa) Relatively bioavailability 21.5% to s.c.

injection. Microemulsion-acceleratedintramucosal transport

Sintov et al., 2010

Thiolated chitosan nanoparticles for improvedmucoadhesion, enzyme inhibition, andpenetration enhancement

Leuprolin (1.2 kDa) Relative bioavailability 19.6%, AUC,Cmax, and elimination half-life improvedby 6.9-, 3.8-, and 4-fold, respectively

Shahnaz et al., 2012a

Cosolvent formulation with n-tridecyl-b-D-maltoside as permeation enhancer in braindelivery

Hexarelin (0.9 kDa) 1.6-fold higher peptide concentration inbrains compared with i.v. administrationdespite lower plasma concentrationsafter intranasal administration

Yu and Kim 2009

PulmonaryHP-b-cyclodextrin and linear or branched

PEG chains spray dried to microparticlesSalmon calcitonin

(3.5 kDa)1.5- and 2.3-fold increase of bioavailability

with branched and linear PEG,respectively, compared with nebulizedsolution

Tewes et al., 2011

Palmityl-acylation of peptide to increaseadsorption on porous PLGA microparticlesand albumin binding in blood circulation

Exendin-4 (4.2 kDa) Native and palmityl-acylated peptideadministered in microparticlesinduced hypoglycemia for 36 h andover 5 days, respectively

Kim et al., 2011

Micellar formulation for improved peptidestability and transepithelial absorption

Salmon calcitonin(3.5 kDa)

1.6-fold increase in bioavailabilityafter administration of micellesthan in a plain solution

Baginski et al., 2012

TransdermalTwo-layered dissolving microneedles prepared

from water-soluble biopolymersDesmopressin

(1.1 kDa)Absolute bioavailability $90% and

maximum plasma concentration reachedin 30 min

Fukushima et al., 2011

Insulin encapsulated nanovesicles deliveredwith iontophoresis through microneedle-induced microchannels

Insulin (5.8 kDa) Comparable hypoglycemic effect on s.c.insulin by using 80-fold higher dose

Chen et al., 2009

Removal surface layers of skin withmicrodermabrasion technique

Insulin (5.8 kDa) Hypoglycemic effect similar to 160-foldlower s.c. insulin dose after removalof epidermis

Andrews et al., 2011

No hypoglycemic effect after removal ofstratum corneum

Peptide Delivery Systems 551

orexin-A (Dhuria et al., 2009), and exendin (Bankset al., 2004). Hexarelin, a growth hormone–releasingpeptide, was delivered to the brain using a cosolventand n-tridecyl-b-D-maltoside for improving the peptidepermeation and enabling 1.6-fold greater plasmaconcentrations compared with intravenous adminis-tration (Yu and Kim, 2009).b. Pulmonary. Lungs are an attractive administra-

tion route for peptides because of their high surfacearea (100–140 m2), high vascularization, and lower enzy-matic activity (Patton, 1996; Dombu and Betbeder,2013). Alveoli also have a thin epithelia layer (0.1–0.5 mm) and no additional mucosal layer, which can allowfast absorption and high bioavailability (10- to 200-foldgreater than in other noninvasive administrationroutes) of pulmonary administered peptides (Pattonand Byron, 2007). Epithelia lining fluid, epithelia celllayer, and endothelia membrane are still formingsignificant absorption barriers, however.Peptides can be administered either as liquid (nebu-

lizator) or as a dry powder, but in both cases, inhalatordevices are needed for efficient pulmonary peptidedelivery. For efficient pulmonary drug delivery, theformulations should have optimal aerodynamic charac-teristics to reach the alveoli, enhance the permeability,prolong the retention time, and control the release of thepeptide and prevent its degradation (Wan et al., 2012).The optimal particle size (mass median aerodynamicdiameter) for alveoli deposition would be 1 to 5 mm,whereas smaller particles (,1 mm) are exhaled, andlarge particles (.10 mm) are deposited in the upperairways (Carvalho et al., 2011). Small nanoparticles(,100 nm) might be also deposited in the alveoli (Mölleret al., 2008; Yang et al., 2008; Geiser and Kreyling 2010).Improved pulmonary bioavailability was seen in

salmon calcitonin microparticles formed by spray dryingHP-b-cyclodextrin with either linear or branched PEGchains (Tewes et al., 2011). Microparticles with linearPEG had lower surface energy and better aerodynamicproperties, whereas branched PEG salmon calcitoninwas more protected from the chemical degradation. Thepulmonary bioavailability of salmon calcitonin micro-particles with branched-PEG was 1.5-fold, and micro-particles with linear-PEG 2.3-fold higher than that ofnebulized salmon calcitonin solution. Absolute bioavail-abilities were 26.3% and 17.0% for linear and branched-PEG, respectively. Another proposed approach forpulmonary delivery of salmon calcitonin was the adsorp-tion of the peptide on the surface of PLGA nanospheres(Yang et al., 2012b). Lyophilized nanospheres werecomposed on inhalable lactose to have micron-sizedparticles to improve inhalation properties. Intratrachealadministration in rats demonstrated that micron-sizedparticles and nanospheres had similar pharmacologiceffects. The third approach for pulmonary delivery ofsalmon calcitonin is micellar formulations that improvethe peptide stability against enzymatic degradation and

increase transepithelial absorption (Baginski et al.,2012). In vivo studies showed 1.6-fold greater bioavail-ability after the administration of salmon calcitonin inmicelles than in plain solution. In 2014, the FDA approvedanother inhaled insulin formulation: MannKind’s Afrezza(Valencia, CA), an inhalable short-acting insulinpowder (http://www.fda.gov/newsevents/newsroom/pressannouncements/ucm403122.htm). Another com-mercially investigated option for inhalable insulinis based on nebulization (Heinemann 2014; http://dancebiopharm.com/dance-501/).

Inhaled delivery can be also used for local delivery ofpeptides needed for treatment of pulmonary diseases. Atthe moment, aviptadil, vasoactive intestinal polypeptide,is in phase 2 clinical studies for treatment of respiratorydistress syndrome and pulmonary hypertension.

c. Transdermal. The easy accessibility of the skinmakes it a desired route for peptide administration, butthe skin barrier needs to be overcome by various chemicaland physical means (Kalluri and Banga 2011; Alexanderet al., 2012; Schoellhammer et al., 2014). The recentapproaches have focused mostly on the iontophoresis andmicroneedle techniques or a combination of the two.

Microneedles are micron-sized, needle-shaped struc-tures that are long enough to penetrate the stratumcorneum but short enough not to evoke pain sensation(van der Maaden et al., 2012). Microneedles offer fourdifferent approaches, all of which have been applied topeptides: 1) pretreatment with microneedles followedby application of the formulation (Zhou et al., 2010;Mohammed et al., 2014); 2) dissolving microneedles thatrelease the peptide rapidly and simultaneously to themicroneedle dissolution (Fukushima et al., 2011; Lingand Chen 2013); 3) coated microneedles releasing theirpeptide coating (Cormier et al., 2004; Tas et al., 2012);and 4) hollow microneedles enabling microinjections(Davis et al., 2005; Wang et al., 2006; Norman et al.,2013). When commercially available microneedle rollerswere used as pretreatment to create micropores into ratskin followed by the application of insulin solution, themicroneedles rollers could increase skin permeability forinsulin-lowering blood glucose levels (Zhou et al., 2010).

Tas et al. (2012) used coated steel microneedles withsalmon calcitonin together with trehalose (for improvedprotein stability) and compared the microneedle admin-istration with subcutaneous injection in rats. The salmoncalcitonin AUC values of microneedle administration andsubcutaneous injection were not significantly different(Tas et al., 2012). Compared with intranasal administra-tion, the AUC after microneedle administration was13-fold higher than after intranasal administration. Twoclinical studies have been performed with parathyroidhormone (1–34)-coated titanium microneedles, and thosestudies showed that transdermal microneedle adminis-tration was able to deliver consistent and therapeuticallyrelevant concentrations (Daddona et al., 2011). The phase2 study even suggested that microneedles could provide

552 Kovalainen et al.

a better plasma profile than subcutaneous injectionbecause of faster absorption. In accordance with thesefindings, Prausnitz and colleagues have shown thatmicroinjections of insulin with hollow microneedles intothe depth of 0.9–1 mm induced a faster onset of insulinaction than subcutaneous injections (Gupta et al., 2009,2011; Norman et al., 2013). This result suggests that themicroneedle injection of insulin is delivered to the pa-pillary dermal region, which has rich capillary andlymphatic networks, allowing faster absorption thanafter subcutaneous injection.Dissolving microneedles can be prepared from various

polymers, and they are usually designed to dissolve inthe skin within minutes to an hour after their applica-tion. In the case of insulin-loaded microneedles, no sig-nificant differences in the blood glucose levels of ratswere found between microneedle and subcutaneousadministrations (Ito et al., 2012; Liu et al., 2012).Hyaluronic acid–based microneedles had a relative phys-iologic activity (RPA) of 94%–98% (Liu et al., 2012),whereas chondroitin sulfate microneedles had a RPA of91%–100% compared with subcutaneous insulin solution(Ito et al., 2012). In dogs, for similar types of chondroitinsulfate microneedles, the RPA value was 59%–71% ofsubcutaneous insulin solution (Fukushima et al., 2010).Dissolving microneedles also have been studied fordelivery of other peptides such as desmopressin with fastabsorption (absorption half-life 14 minutes) and highbioavailability (90%–93%) (Fukushima et al., 2011).Peptides were chemically stable for at least 1 month ofstorage at +4°C (Fukushima et al., 2010, 2011) or at roomtemperature (Ito et al., 2012; Liu et al., 2012).Iontophoresis uses small currents to drive charged

molecules through the skin and has been studied forpeptide delivery (Herwadkar and Banga, 2012). Peptidesfor iontophoretic delivery must be charged and ideallyhave their isoelectric point either below 4 or above 7.4.Chen et al. (2009) circumvented this requirement byencapsulating insulin (isoelectric point 5.4) into chargednanovesicles that masked the charge of insulin. Accord-ing to the authors, nanovesicles themselves were able topenetrate through the skin, but iontophoresis increasedpenetration by 3.3- to 5.3-fold. The combination ofiontophoresis with micropores created by microneedlesincreased penetration rate by 93- to 145-fold. Thesynergetic effects of iontophoresis and microneedle-created micropores are based on the ability of iontopho-resis to increase transport and micropores to decreasethe skin barrier properties. Similarly, the combination ofmicroneedles and iontophoresis increased maximumplasma concentration of salmon calcitonin 9-fold incomparison with iontophoresis alone, and therapeuticlevels were achieved within 5 minutes (Vemulapalliet al., 2012).Other techniques for transdermal peptide delivery in

vivo include cell-penetrating peptides (Chang et al.,2013), microdermabrasion (Andrews et al., 2011), and

electroporation (Wong et al., 2011). Chang et al. (2013)synthesized and screened 20 cationic cyclopeptidesbased on highly hydrophilic cyclic peptides (namedTD-1; ACSSSPSKHCG). Cyclopeptide, a cell-penetratingpeptide with bisubstituted lysine, was the mosteffective in lowering blood glucose levels and looseningof epidermal tight junctions. In microdermabrasion,the top skin layers, namely, the stratum corneum andepidermis, can be locally removed to enhance thedelivery of peptides. In the case of insulin, removal ofboth stratum corneum and epidermis had similarglucose-lowering effects than subcutaneous injection(Andrews et al., 2011). The removal of only the stratumcorneum had no effect on insulin absorption, and it wasconcluded that not only stratum corneum but alsoepidermis form an absorption barrier for insulin.Electroporation creates temporary structural pertur-bation of lipid membrane bilayers by using very shorthigh-voltage pulses (Alexander et al., 2012). Electro-poration of insulin, particularly when combined withlocal hyperthermia, has been shown to lower bloodglucose levels, although no subcutaneous insulin in-jection was used as a control (Wong et al., 2011).

IV. Porous Silicon as a Novel Material forPeptide Delivery

The interest in investigating porous silicon (PSi) asa drug carrier has arisen from promising features of thisbiocompatible material, which include the ability to 1)carry large drug amounts; 2) enhance the dissolution ofdrugs with low solubility; and, most important, and 3)have its properties modified (Anglin et al., 2008;Salonen et al., 2008; Jaganathan and Godin, 2012).PSi differs from many of the commonly used drugcarriers since the loading of peptides is much easiercompared with, for example, typically used polymermaterials, and there is no need to use conditions thatcan be harmful for peptides such as strong solvents orhigh temperatures. Our research group demonstratedthe promising features of PSi for sustained delivery ofpeptides (Kilpeläinen et al., 2009; Kovalainen et al.,2012; Huotari et al., 2013). In the following sections, wegive an overview on the PSi material and its use inpeptide delivery where the subcutaneous route hasshown to be the preferable administration route.

A. Fabrication and Properties of Porous Silicon

PSi consists of pore walls of elemental silicon withhighly tunable morphologies (Fig. 3). PSi has beenknown for almost six decades, and during this time, theperception of the material has evolved from an un-wanted side product to a scientific curiosity, a potentialmaterial in electronics, and, more recently, in drugdelivery systems. This fascinating material combinesthe properties of nanomaterials, semiconductors, and

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biomaterials, and it has therefore many beneficialqualities to be exploited in various applications.The oldest and still most commonly used method to

produce PSi is electrochemical anodization. Already in1956, Uhlir (1956) had observed that the surface ofsilicon wafer changed its color if it was anodized with anappropriate current density in an electrolyte solutioncontaining hydrofluoric acid. In anodization, siliconserves as an anode electrode, and the cathode is usuallymade of platinum. When a certain etching current isconducted through the electrodes, a porous layer isformed on the surface of the silicon substrate. In the film,the pores typically pass perpendicularly through the film,which can be detached from the substrate and processedfor further use. Several different types of pore structurescan be produced by electrochemical etching: from ran-domly orientated, spongelike pore structures to highlyordered cylindrical pores with smooth pore walls andpore sizes from a few-nanometers-wide micropores tomicrometer-scale macropores (Heinrich et al., 1992;Salonen et al., 2005; Bimbo et al., 2010). The controlover particle size is especially important when consider-ing different administration routes.Crystalline silicon has long been considered as bioinert

material, until, in 1995, PSi was found to be bioactive(Canham, 1995), with its biologic behavior dependent onits porosity (Bowditch et al., 1999; Canham et al., 2000;Anderson et al., 2003). PSi dissolves to orthosilicic acid,a natural and biologically important form of silicon, forexample, for optimal bone and collagen growth (Andersonet al., 2003; Salonen et al., 2008). Another beneficialproperty of PSi is the possibility to make it photo-luminescent (Canham, 1990). In biomedical applications,the particles can be tracked by fluorescent microscopywithout additional fluorescent labels that can cause un-desired changes in the surface chemistry (Gu et al., 2013).

B. Porous Silicon as a Peptide Delivery System

Controlling surface chemistry of PSi is highlyimportant in the drug delivery application. Surfacechemistry has three basic functions for the material: 1)it determines the rate of degradation, together with

porosity; 2) it controls interactions between PSi andthe loaded molecules; and 3) it determines the inter-actions of PSi with biologic systems.

PSi surface is typically modified in two steps. First, thenative unstable hydrogen terminated surface of PSi isreplaced with a passivation layer to improve its stabilityin biologic environments. Second, the stabilized surface isfunctionalized with molecules that typically have func-tional carboxyl or amine groups. These functional groupsdetermine the particle surface charge, which may befurther modified, for example, with biologically activemolecules.

The most frequently used method of stabilizing PSi isoxidation (Riikonen et al., 2012). This can be performedin gas phase at elevated temperatures (250–1000°C) orin liquid phase with oxidants such as hydrogen per-oxide. The formed silicon oxide surface is hydrophilicwith an intermediate stability and has 2OH groups onthe surface for further functionalization.

Carbonization is an effective stabilization method thatcreates surfaces more stable than the oxide surfaces. Itcan be performed by thermal decomposition of carbon-containing molecules such as acetylene or poly(furfuryl)alcohol on hydrogen terminated PSi surface (Salonenet al., 2002, 2004; Tsang et al., 2012). At relatively lowtemperatures (i.e., around 500°C), acetylene gas canpartially decompose and form a hydrocarbon terminatedsurface on PSi. This thermal hydrocarbonization processproduces relatively stable surfaces with hydrophobiccharacteristics. Even more stable surfaces can be form-ed by thermal carbonization at higher temperatures(i.e., around 800°C), at which the carbon atoms can enterthe crystal structure of PSi and form a silicon carbidesurface. These surfaces are more hydrophilic and highlyresistant to various chemical environments.

Hydrogen terminated PSi surfaces can be directlyfunctionalized by hydrosilylation (Buriak, 2002). In thismethod, a molecule is grafted on the PSi surface bya reaction between terminal carbon-carbon double bondof the grafted molecule and a hydride groups on the PSisurface. Various alkene molecules can be grafted on PSisurface by this method. It also stabilizes the surface tosome extent compared with unmodified PSi surface,especially if the grafted molecules are highly hydrophobic(Buriak and Allen, 1998). A similar method has recentlybeen used to graft undecylenic acid on thermally hydro-carbonized PSi (Jalkanen et al., 2012).

Oxidized or carbonized surfaces can be functionalizedby silanization, in which alkoxysilanes react with 2OHgroups on the stabilized surface (Mäkilä et al., 2012; Xuet al., 2012). This is the most commonly used function-alization method of silica-based materials, and it is mostoften used to attach amine terminated molecules on thesurface. These surfaces have a positive charge in phys-iologically relevant solutions and may need to be furthermodified for biomedical use. Because of the positive chargeand reactivity of amine groups, these amine-terminated

Fig. 3. Scanning electron microscopy image of peptide loaded mesopo-rous silicon particles (38–53 mm) and porous structure (pore diameterabout 15 nm).

554 Kovalainen et al.

surface modifications are the only surface chemistry thathas shown some cytotoxic effects during in vitro studies.All the other stabilized surface chemistries have beenfound to be nontoxic at the clinically relevant concen-trations (Lehto et al., 2013).To provide more advanced biologic properties, PSi

needs to be further functionalized. Targeting peptidescan be grafted on the surface to enhance accumulationPSi nanoparticles into target tissue (Kinnari et al., 2013;Yokoi et al., 2013). The pores can also be capped withproteins or cyclodextrin, which enable the release of theloaded molecules at certain pH (Perelman et al., 2008;Xue et al., 2011). Furthermore, PSi particles can beencapsulated by, for example, solid lipids to improvetheir behavior in vivo (Liu et al., 2013b). In a recentstudy, PSi particles were covered with cellular mem-branes of leukocytes, giving the particles advanced cell-like functions (Parodi et al., 2013).When considering peptide loading in a drug carrier, it

is important that the loading method be as simple andas gentle as possible to preserve the bioactivity of thepeptides. This is a clear advantage of PSi compared withmany other drug delivery systems in which the payloadhas to be added into the fabrication process of thedelivery system. In the case of PSi, it is possible to loadthe drug afterward, at room temperature, using mildsolvents.The drug-loading methods of PSi can be divided into

two categories. In immersion loading, PSi is immersed ina loading solution, the volume of which is clearly greaterthan the pore volume of the PSi. In many cases, thisrequires excess of drug to obtain high payload to carriermass ratio in the system. Usually, the method alsorequires removal of PSi from the loading solution. Thiscan be done by filtration; or, in the case of nanoparticles,centrifugation is preferred. The other category of loadingmethods is so-called impregnation (incipient wetnessmethod). In this type of method, the amount of drugsolution corresponding to the pore volume of PSi is addedto the sample and allowed to infuse into the pores bycapillary action. The advantage of this method is the highloading efficiency, minimizing the wasted drug. On theother hand, because of the low volume of loading solution,the payload (loading degree) obtained can be rather low.Crystallization of the drug on the external surface of PSiparticles (outside the pores) can occur; however, bothmethods are performed at room temperature, and vir-tually any solvent suitable for the peptide can be used.The loading is simple to perform, but the interactions

involved in the loading are more difficult to control. Inmost cases, chemical reactions between the drug mol-ecules and the pore wall are undesired and can beavoided with the proper choice of the surface chemistryof PSi. These interactions (e.g., drug-solvent, drug-porewall, and solvent-pore) affect the loading degree. De-termination of the drug-loading degree is determined byliquid extraction of the drug and measurement is by

high-performance liquid chromatography; the amount ofdrug in PSi can be determined using thermogravimeter(Lehto et al., 2005) (Fig. 4).

Kovalainen et al. (2012) studied the loading efficiencyof PYY3-36 in PSi with three different surface chemistries(Fig. 5). Interestingly, loading efficiency (i.e., whatpercentage of the drug from the solution is loaded intothe pores) close to 100% was observed in the case ofthermally hydrocarbonized (hydrophobic) PSi withgreater than 7 w-% loading degree. Similar behaviorwas observed with splice correction oligonucleotideswhere greater than 14 w-% loading degree was obtainedwith positively charged PSi nanoparticles, the loadingefficiency being 100% (Rytkönen et al., 2014). Thishighlights the ability of PSi to adsorb all the drugmolecules from the solution in suitable conditions. Thesurface chemistry plays an important role in the releaseand bioavailability of subcutaneously administratedpeptide (Kovalainen et al., 2012).

The first study in which PSi silicon has been used invitro with peptides or proteins was reported by Forakeret al. (2003). In their article, oxidized PSi was loadedwith insulin, and the permeability of insulin throughCaco-2 cell membrane was observed to increase due tothe PSi microparticles. Prestidge and coworkers havesystematically studied interactions between peptides/proteins and PSi (Prestidge et al., 2008; Jarvis et al.,2010, 2012), and how various parameters like surfacemodifications, together with pore diameters, affect theloading degree. They have also shown that surface affectshow peptides are adsorbed on PSi surfaces. The firstdemonstration of using PSi for peptide delivery in vivoshowed that PSi microparticles prolong the pharmacody-namics response of a food intake regulating peptides inmice (Kilpeläinen et al., 2009). Later investigation of

Fig. 4. Results of thermogravimetric measurements of peptide ghrelinantagonist (GhA) and loaded into PSi microparticles. The results shownear complete decomposition of GhA as such. The GhA-loaded PSimicroparticles show a mass loss of 18.5%, which can also be assumed asthe loading degree of the particles.

Peptide Delivery Systems 555

peptide pharmacokinetics proved that controlled releaseover several days can be achieved (Kovalainen et al.,2012).In addition, PSi microparticles were used to carry

antigenic peptides into human monocyte–derived den-dritic cells to enhance the generation of antiviral cytotoxicT-lymphocyte response (Jimenez-Perianez et al., 2013).PSi appears to be a potential peptide delivery system

as a result of its several advantageous properties, likebioresorbability, nontoxicity, high peptide-loading ca-pacity, and easy physicochemical modification of theconstituent. PSi has also been shown to be capable tocarry different molecules simultaneously (Liu et al.,2013a). Loading capacity determining factors fromprevious studies are summarized in Table 8.Biodegradation of PSi has not been evaluated system-

atically. PEGylation of the chemically oxidized PSiparticles was recognized to prolong the degradation timeof PSi by 3 days (Godin et al., 2010). Biodegradation invivo has been evaluated for oxidized PSi particles whendelivered intravenously, but a corresponding study hasnot been conducted for subcutaneously injected particles(Park et al., 2009). It is known that the injected particlesdo not migrate from the deposit site for 4 hours (Bimboet al., 2010), but after 4 weeks, thermally hydrocarbon-ized microparticles have mostly dissolved from theinjection site. As the peptide release can be modulatedby changing the biodegradation rate of the carrier, and asthe in vivo–in vitro correlation is known to be poor in this

regard, knowledge about the real degradation rates aftersubcutaneous delivery would be critical for the develop-ment of PSi formulation for sustained peptide delivery.

V. Immunogenicity and Adverse Effects ofParenteral Peptide Delivery Systems

A. Immunogenicity

Risk of immunogenic reactions exists when biologiccompounds are used as drugs, and the severity of thereaction may vary from negligible events to seriousanaphylactic shock (Vugmeyster et al., 2012). Particu-larly, compounds that are originated from other speciesthan humans tend to stimulate formation of anti-bodies. As an example, the original insulin formula-tions from bovine and porcine pancreas induced moreallergic reactions compared with insulins with humanamino acid sequence. Formation of antidrug antibodiesmay alter the ADME and pharmacodynamics, whichmay result in enhanced clearance or altered biologicactivity of the drug (Vugmeyster et al., 2012).

B. Adverse Effects

The use of particulate delivery systems in medicineresults in direct exposures of body cells and tissues toforeign materials. Physicochemical properties (size,shape, surface chemistry, and charge), along with thesite of the particle’s administration, define their protein

Fig. 5. Surfaces of PSi can be modified in various ways that affect interactions between the peptide and the carrier (e.g., via electrostatic interactionand hydrophobic interaction). The figure presents three surface modifications that were studied with peptide YY (Kovalainen et al., 2012): a) thermallyoxidized Psi, b) thermally hydrocarbonized Psi, and c) carboxylated thermally hydrocarbonized PSi.

TABLE 8Factors determining the drug-loading capacity of PSi

The surface area is an essential parameter to obtaining high loading capacity/degree.The ratio of the pore diameter to the peptide molecule size needs to be sufficiently high to achieve effective penetration of the molecules in the pores.Hydrophobic surfaces can facilitate peptide loading in aqueous solutions but might be hazardous to peptide stability if the surface of PSi is not passivized before the

peptide loading.Electrostatic attraction is an effective way to promote loading of the peptides in PSi carriers.The charges of the peptide and PSi particles depend on the properties of the medium (e.g., solvent, pH, and excipients), which need to be considered in planning the loading

procedure.

556 Kovalainen et al.

and cellular interactions (Nel et al., 2009; Arora et al.,2012; Treuel et al., 2013). Changing the PSi particlesurface characteristics offers unique advantages todesign a carrier that can efficiently deliver therapeuticmolecules to diseased tissue (Limnell et al., 2007;Jarvis et al., 2011). Yet changing of the particle’s verysame properties can result in their unexpected bio-interactions and adverse effects. Understandingwhether the drug carriers themselves may induceadverse effects is important for engineering safe-in-usedelivery systems.Size is an important property for the development of

parenteral and especially for intravenous deliverysystems. The recommended size of the particles forintravenous administration should not exceed thediameter of the fine capillaries (9 mm), preferentiallystaying in submicron range to avoid their possibleblockage, which could lead to a fat embolism and resultin death (Mehnert and Mader, 2001). This cutoff is themost critical for the particles with limited deform-ability or particles tending to aggregate, such as lipid-based drug carriers (Elgart et al., 2012).Reduction of the particle size to nanoscale generates

extremely high surface area to volume ratio, whichdetermines an increase in the potential number of thereactive groups on the particles surface and creates theopportunity for their increased uptake, intense interac-tion with and translocation through the biologic tissues.Therefore, a smaller range size can became a key factorof nanoparticle’s safety. Attachment of nanoparticles tothe surface of the red blood cells (RBCs) has beensuggested as a tool for extending circulation time andsustained release of therapeutics (Hall et al., 2007). Ithas recently been found that this surface modification ofnanoparticles can size-dependently decrease membranedeformability of RBCs, which is critical for effective bloodflow (Zhao et al., 2011). This study revealed that amongseveral surface types and sizes of PSi nanoparticles, onlysmall MCM-41–type nanoparticles in a range of 100–200 nm can adsorb to the RBC surface without affectingtheir membrane or morphology; however, the interac-tions of these nanoparticles with other blood cells shouldbe evaluated before the final conclusion about theirhemocompatibility is made (Semberova et al., 2009).Design of charged nanoparticles is gaining popularity

in drug delivery applications (Malik et al., 2000; Elgartet al., 2012; Lin et al., 2013). Modulation of the surfacecharge is used to stabilize colloidal systems of nano-particles (Elgart et al., 2012), to prolong the circulationtime (Malik et al., 2000), and to increase cellular uptake(Lin et al., 2013). At the same time, the charge can bethe critical factor for electrically excitable cardiac tissue.Recently, it has been shown that polysterene latexnanoparticles, which were amine-modified and havea positive charge, induced large-scale damage tocardiomyocytes, leading to cell death (Miragoli et al.,2013). By contrast, negatively charged nanoparticles of

the same nature (carboxyl-modified polystyrene latexnanoparticles) were not cytotoxic; however, exposure tonegatively charged nanoparticles changed electrophys-iologic characteristics of cardiomyocytes, sensitizingthem toward arrhythmias (Miragoli et al., 2013).

In conclusion, it is not possible to define any newchemical material as nontoxic without testing it in thefinal application in vivo. The diversity of the particles,in particular nanosized particles, being developed asadvanced delivery systems makes prediction of theirpossible adverse effects according to physicochemicalcharacteristics quite difficult. The increasing volume indata on nanotoxicology, entailing physicochemical deter-minants, biodistribution, and biologic effects of nano-particles defines the key points for testing their toxicity.Thus, the risk of in vivo use of nanoparticle deliverysystems should be thoroughly assessed by combination ofthe test procedures on case-by-case basis.

VI. Future Potential of Peptide Drugs

The predicted forecast for the future drug discoveryis that more and more potential drug targets involveprotein-protein interactions. An additional challengein the future will be cell-penetrating peptides, whichtarget intracellular proteins.

The market for protein- and peptide-based drugs iscurrently estimated at greater than $40 billion peryear, or 10% of the total pharmaceutical market. Thismarket share is growing much faster than that of otherpharmaceutical fields, and success rates for bringingbiologics to market are now about twice that of small-molecule drugs (Craik et al., 2013). Although thefigures presented for biologicals refer mainly refer toblockbuster monoclonal antibodies, it can be predictedthat the share of peptidergic drugs will increase aswell. Kaspar and Reichert (2013) analyzed the sales ofthe 25 top peptide therapeutics and found that theglobal sales accumulated to $14.7 billion in 2011. Thiswill put pressure on manufacturing technology as wellas on innovative delivery and transport technologies.

The list of bioactive peptides with potential thera-peutic value is huge and is growing each year (Kasparand Reichert, 2013). More and more potential thera-peutic lead peptides can be found by proteomics andscreening of natural sources, like animal venoms,which are known to be potent and fast acting. In thefuture, as the biologic therapeutic segment grows,peptides may appear not only as prescribed drugs, butas functional foods and nutraceuticals. There is alsogreat potential in peptide antigens in vaccines anddiagnostics. It can be predicted that peptidergic drugswill be emerging as an important therapeutic alterna-tive to large biologic and small-molecule drugs.

Acknowledgments

The authors thank Dr. Dominique Gagnon for spell checking andDr. Kari Mäkelä for assisting with the figures.

Peptide Delivery Systems 557

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Kovalainen,Mönkäre, Riikonen, Pesonen, Vlasova, Salonen, Lehto, Järvinen,Herzig.

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